PI-kinase Inhibitors with Broad Spectrum Anti-Infective Activity

ABSTRACT

Compounds and methods are provided for the treatment of pathogen infections. In some embodiments, the anti-infective compounds have broad spectrum activity against a variety of infective diseases, where the diseases are caused by pathogens containing a basic amino acid PIP-2 pincer (BAAPP) domain that interacts with phosphatidylinositol 4,5-bisphosphate (PIP-2) to mediate pathogen replication. Also provided are methods of inhibiting a PI4-kinase and methods of inhibiting viral infection. In some embodiments, the compound is a PI4-kinase inhibiting compound that is a 5-aryl-thiazole. The subject compounds may be formulated or provided to a subject in combination with a second anti-infective agent, e.g. interferon, ribivarin, and the like.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/543,538,filed Oct. 5, 2011.

GOVERNMENT RIGHTS

This invention was made with Government support under grants T32DK007056, T32 AI007502-14 awarded by the National Institutes of Health.The Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention pertains to methods and compounds for treatment ofpathogen infections.

BACKGROUND

The rapid rise in the number of emerging pathogens in the world'spopulation represents a serious global health problem and underscoresthe need to develop broad spectrum anti-infectives that target commoncomponents of large classes of pathogens.

For example, it is estimated that more than 2% of the world's populationis currently infected with the Hepatitis C Virus (HCV). One of theoutstanding characteristics of HCV is its ability to establish chronicinfections in 65-80% of infected patients. Chronic infection with HCVcan lead to serious sequelae including chronic active hepatitis,cirrhosis and hepatocellular carcinoma—usually manifested 10, 20 and 25years respectively after the initial infection. End stage liver diseasefrom HCV has become the leading indication for liver transplantation inNorth America, and it has been suggested that there will be a 2-3 foldincrease in liver transplantation in 10 years as a result of cirrhosisfrom hepatitis C.

Broad spectrum anti-infective agents for use in treating variousinfective diseases are of interest. Also of interest are anti-infectiveagents for specifically treating one or more pathogen caused infectivediseases, such as HCV.

SUMMARY OF THE DISCLOSURE

Compounds and methods are provided for the treatment of pathogeninfections, which include, without limitation, viruses and otherpathogens that utilize intracellular replication mechanisms, e.g.hepatitis C virus (HCV), Plasmodium falciparum, rhinovirus, and thelike. In some embodiments, the anti-infective compounds have broadspectrum activity against a variety of infective diseases, where thediseases are caused by pathogens containing a basic amino acid PIP2pincer (BAAPP) domain that interacts with phosphatidylinositol4,5-bisphosphate (PI(4,5)P₂) to mediate replication.

Also provided are methods of inhibiting a PI4-kinase and methods ofinhibiting viral infection in a subject. In some embodiments, thecompound is a PI4-kinase inhibiting compound that is a 5-aryl-thiazole,e.g., as described herein. In certain embodiments the compound is a2-amino-5-phenylthiazole compound. The subject compounds may beformulated or provided to a subject in combination with one or moreadditional anti-infective agents, e.g. interferon, ribavirin, and thelike. For treatment of viruses such as HCV, the compounds may beformulated to specifically target the liver, e.g. by conjugation withpolyarginine or a bile acid, or as pro-drugs designed to be activated byenzymes resident in the liver.

These and other advantages, and features of the disclosure will becomeapparent to those persons skilled in the art upon reading the details ofthe compositions and methods of use, which are more fully describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that the exemplary compound PT423 decreases PI(4)P(top panels) and PI(4,5)P₂ (bottom panels) in a dose-dependent manner.Left panels: uninfected Huh7.5 cells; right panels: HCV-infected Huh7.5cells. Cells were treated with the indicated concentrations of PT423 orvehicle control, and analyzed by immunofluorescence with antibodies toPI(4)P or PI(4,5)P₂ (green) along with an antibody to calnexin (red) tocontrol for ER morphology.

FIG. 2 illustrates the major metabolites identified for the exemplarycompound PT423 in a microsome assay.

DEFINITIONS

Before embodiments of the present disclosure are further described, itis to be understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Any methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of embodiments of the present disclosure.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes not only a single compound but also a combination oftwo or more compounds, reference to “a substituent” includes a singlesubstituent as well as two or more substituents, and the like.

In describing and claiming the present invention, certain terminologywill be used in accordance with the definitions set out below. It willbe appreciated that the definitions provided herein are not intended tobe mutually exclusive. Accordingly, some chemical moieties may fallwithin the definition of more than one term.

As used herein, the phrases “for example,” “for instance,” “such as,” or“including” are meant to introduce examples that further clarify moregeneral subject matter. These examples are provided only as an aid forunderstanding the disclosure, and are not meant to be limiting in anyfashion.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The Basic Amino Acid PIP2 Pincer (BAAPP) domain, as described by Glennet al., “PIP-2 Inhibition-Based Antiviral and Anti-HyperlipidemicTherapies” WO2009/148541, and which is herein incorporated by referencein its entirety, provides a mechanism by which a protein or peptiderecognizes (including but not limited to binding, as well as activationor suppression of activity) PIP2 (phosphatidylinositol 4,5-bisphosphate[PtdIns(4,5)P2], PI(4,5)P2). Alterations or variations of the BAAPPdomain may result in recognition of other phosphatidylinositol variants.

Phosphoinositides, such as phosphatidylinositol (PI)-4-phosphate(PI(4)P) and PI-4,5-bisphosphate (PI(4,5)P₂, or “PIP2”), are enriched invarious specific plasma membrane and intracellular locations. The steadystate location and abundance of specific PI isoform pools within thecell is regulated by a family of PI-kinases and phosphatases. There areleast 4 human PI4-kinases, with family members PI4KIIIα and PI4KIIIβbeing primarily localized to ER and Golgi-derived membranes where theycontribute to the PI(4)P and PI(4,5)P₂ pools associated with thesemembranes, and with family members PI4KIIα and PI4KIIIβ contributingprimarily to other pools.

The BAAPP domain mediates specific interaction with PIP2, resulting in aconformational change in the BAAPP domain that affects a key pathogenregulator. In HCV, replication complexes are established atintracellular PIP2-enriched sites, and point mutations in the BAAPPdomain abrogate PIP2 binding and HCV RNA replication. Such criticaldependence on PIP2 is widespread among pathogens. Targeting specificintracellular PIP2 pools by siRNA-mediated knockdown of enzymesresponsible for PIP2 production—such as PI4KIIIα and PI4KIIIβ—abrogatesHCV replication, yet is well tolerated by the host cell.

Molecules that inhibit the enzymatic pathways responsible for productionof PIP-2 are of interest for use in the methods of the disclosure. Suchinhibitors include, without limitation, inhibitors ofphosphatidylinositol 4-kinase III alpha (see, for example Berger et al.(2009) PNAS 106:7577-7582, herein specifically incorporated byreference) and inhibitors of phosphatidylinositol 4-kinase III beta.

BAAPP domains have been identified in multiple organisms, including butnot limited to pathogens such as viruses, bacteria, fungi and parasites,as well as hosts, such as the human. BAAPP domain peptides, moleculesthat mimic the BAAPP domain, enzymes involved in PIP-2 metabolism, andmolecules that inhibit or activate the BAAPP domain act in treatinginfectious diseases as well as affecting host physiology orpathophysiology.

Examples of proteins having a BAAPP domain include, without limitation,the 2C protein of Picornaviridae, Rhinovirus 14, Rhinovirus B,Rhinovirus C, PolioVirus, Enterovirus A, Enterovirus B, Enterovirus C,Enterovirus D, Enterovirus 71, and Coxsackie A virus 18. The coreprotein of Japanese Encephalitis Virus, West Nile Virus, Dengue Virus 1,Dengue Virus 2, Dengue Virus 3, and Dengue Virus 4 have BAAPP domains,as does the P. falciparum PfNDH2 protein. In the Flaviviridae, the NS4BAH 1 of HCV; the NS5A protein of HCV which has a BAAPP domain thatcomprises the conserved lysine residues at residue 20 and 26 of theprocessed protein, for example a peptide with the amino acid sequenceSGSWLRDVWDWICTVLTDFKTWLQSKLL (SEQ ID NO:1) that includes the lysineresidues K20 and K26. Other BAAPP-domain harboring pathogens includeHAV, Vaccinia, Ebola virus, F. Tularensis, influenza virus polymeraseprotein, Variola major (smallpox), Sin Nombre virus (hantavirus),Pseudomonas aeruginosa, CMV.

NS5 encoding viruses include without limitation flaviviruses,pestiviruses and hepatitis C viruses, e.g. yellow fever virus (YFV);Dengue virus, including Dengue types 1-4; Japanese Encephalitis virus;Murray Valley Encephalitis virus; St. Louis Encephalitis virus; WestNile virus; tick-borne encephalitis virus; Hepatitis C virus; Kunjinvirus; Central European encephalitis virus; Russian spring-summerencephalitis virus; Powassan virus; Kyasanur Forest disease virus; andOmsk hemorrhagic fever virus.

By “Flaviviridae virus” is meant any virus of the Flaviviridae family,including those viruses that infect humans and non-human animals. Thepolynucleotide and polypeptide sequences encoding these viruses are wellknown in the art, and may be found at NCBI's GenBank database, e.g., asGenbank Accession numbers NC_(—)004102, AB031663, D11355, D11168,AJ238800, NC_(—)001809, NC_(—)001437, NC_(—)004355 NC_(—)004119,NC_(—)003996, NC_(—)003690, NC_(—)003687, NC_(—)003675, NC_(—)003676,NC_(—)003218, NC_(—)001563, NC_(—)000943, NC_(—)003679, NC_(—)003678,NC_(—)003677, NC_(—)002657, NC_(—)002032, and NC_(—)001461, the contentsof which database entries are incorporated by references herein in theirentirety.

The terms “active agent,” “antagonist”, “inhibitor”, “drug” and“pharmacologically active agent” are used interchangeably herein torefer to a chemical material or compound which, when administered to anorganism (human or animal) induces a desired pharmacologic and/orphysiologic effect by local and/or systemic action.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect, such asreduction of viral titer. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete cure for a diseaseand/or adverse affect attributable to the disease. “Treatment,” as usedherein, covers any treatment of a disease in a mammal, particularly in ahuman, and includes: (a) preventing the disease or a symptom of adisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it (e.g., includingdiseases that may be associated with or caused by a primary disease (asin liver fibrosis that can result in the context of chronic HCVinfection); (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease(e.g., reduction in viral titers).

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to an animal, including, but notlimited to, human and non-human primates, including simians and humans;rodents, including rats and mice; bovines; equines; ovines; felines;canines; and the like. “Mammal” means a member or members of anymammalian species, and includes, by way of example, canines; felines;equines; bovines; ovines; rodentia, etc. and primates, e.g., non-humanprimates, and humans. Non-human animal models, e.g., mammals, e.g.non-human primates, murines, lagomorpha, etc. may be used forexperimental investigations.

As used herein, the terms “determining,” “measuring,” “assessing,” and“assaying” are used interchangeably and include both quantitative andqualitative determinations.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and native leader sequences, with or withoutN-terminal methionine residues; immunologically tagged proteins; fusionproteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, control regions, isolated RNA ofany sequence, nucleic acid probes, and primers. The nucleic acidmolecule may be linear or circular.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, condition, or disorder, is sufficient toeffect such treatment for the disease, condition, or disorder. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the subjectto be treated.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a compound(e.g., an aminopyrimidine compound, as described herein) calculated inan amount sufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for unit dosage forms depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, diluent, carrier andadjuvant” as used in the specification and claims includes both one andmore than one such excipient, diluent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and preferably free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous,and the like.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used. The term “independentlyselected from” is used herein to indicate that the recited elements,e.g., R groups or the like, can be identical or different.

As used herein, the terms “may,” “optional,” “optionally,” or “mayoptionally” mean that the subsequently described circumstance may or maynot occur, so that the description includes instances where thecircumstance occurs and instances where it does not. For example, thephrase “optionally substituted” means that a non-hydrogen substituentmay or may not be present on a given atom, and, thus, the descriptionincludes structures wherein a non-hydrogen substituent is present andstructures wherein a non-hydrogen substituent is not present.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group (i.e., a mono-radical) typically althoughnot necessarily containing 1 to about 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl,and the like, as well as cycloalkyl groups such as cyclopentyl,cyclohexyl and the like. Generally, although not necessarily, alkylgroups herein may contain 1 to about 18 carbon atoms, and such groupsmay contain 1 to about 12 carbon atoms. The term “lower alkyl” intendsan alkyl group of 1 to 6 carbon atoms. “Substituted alkyl” refers toalkyl substituted with one or more substituent groups, and this includesinstances wherein two hydrogen atoms from the same carbon atom in analkyl substituent are replaced, such as in a carbonyl group (i.e., asubstituted alkyl group may include a —C(═O)— moiety). The terms“heteroatom-containing alkyl” and “heteroalkyl” refer to an alkylsubstituent in which at least one carbon atom is replaced with aheteroatom, as described in further detail infra. If not otherwiseindicated, the terms “alkyl” and “lower alkyl” include linear, branched,cyclic, unsubstituted, substituted, and/or heteroatom-containing alkylor lower alkyl, respectively.

The term “alkenyl” as used herein refers to a linear, branched or cyclichydrocarbon group of 2 to about 24 carbon atoms containing at least onedouble bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl,isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl,tetracosenyl, and the like. Generally, although again not necessarily,alkenyl groups herein may contain 2 to about 18 carbon atoms, and forexample may contain 2 to 12 carbon atoms. The term “lower alkenyl”intends an alkenyl group of 2 to 6 carbon atoms. The term “substitutedalkenyl” refers to alkenyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkenyl” and“heteroalkenyl” refer to alkenyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkenyl” and “lower alkenyl” include linear, branched, cyclic,unsubstituted, substituted, and/or heteroatom-containing alkenyl andlower alkenyl, respectively.

The term “alkynyl” as used herein refers to a linear or branchedhydrocarbon group of 2 to 24 carbon atoms containing at least one triplebond, such as ethynyl, n-propynyl, and the like. Generally, althoughagain not necessarily, alkynyl groups herein may contain 2 to about 18carbon atoms, and such groups may further contain 2 to 12 carbon atoms.The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbonatoms. The term “substituted alkynyl” refers to alkynyl substituted withone or more substituent groups, and the terms “heteroatom-containingalkynyl” and “heteroalkynyl” refer to alkynyl in which at least onecarbon atom is replaced with a heteroatom. If not otherwise indicated,the terms “alkynyl” and “lower alkynyl” include linear, branched,unsubstituted, substituted, and/or heteroatom-containing alkynyl andlower alkynyl, respectively.

The term “alkoxy” as used herein intends an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group may berepresented as —O-alkyl where alkyl is as defined above. A “loweralkoxy” group intends an alkoxy group containing 1 to 6 carbon atoms,and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy,t-butyloxy, etc. Substituents identified as “C₁₀₆ alkoxy” or “loweralkoxy” herein may, for example, may contain 1 to 3 carbon atoms, and asa further example, such substituents may contain 1 or 2 carbon atoms(i.e., methoxy and ethoxy).

The term “aryl” as used herein, and unless otherwise specified, refersto an aromatic substituent generally, although not necessarily,containing 5 to 30 carbon atoms and containing a single aromatic ring ormultiple aromatic rings that are fused together, directly linked, orindirectly linked (such that the different aromatic rings are bound to acommon group such as a methylene or ethylene moiety). Aryl groups may,for example, contain 5 to 20 carbon atoms, and as a further example,aryl groups may contain 5 to 12 carbon atoms. For example, aryl groupsmay contain one aromatic ring or two or more fused or linked aromaticrings (i.e., biaryl, aryl-substituted aryl, etc.). Examples includephenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone,and the like. “Substituted aryl” refers to an aryl moiety substitutedwith one or more substituent groups, and the terms“heteroatom-containing aryl” and “heteroaryl” refer to aryl substituent,in which at least one carbon atom is replaced with a heteroatom, as willbe described in further detail infra. Aryl is intended to include stablecyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturatedC₃-C₁₄ moieties, exemplified but not limited to phenyl, biphenyl,naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, andoxazoyl; which may further be substituted with one to five membersselected from the group consisting of hydroxy, C₁-C₈ alkoxy, C₁-C₈branched or straight-chain alkyl, acyloxy, carbamoyl, amino,N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (seee.g. Katritzky, Handbook of Heterocyclic Chemistry). If not otherwiseindicated, the term “aryl” includes unsubstituted, substituted, and/orheteroatom-containing aromatic substituents.

The term “aralkyl” refers to an alkyl group with an aryl substituent,and the term “alkaryl” refers to an aryl group with an alkylsubstituent, wherein “alkyl” and “aryl” are as defined above. Ingeneral, aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms.Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbonatoms, and as a further example, such groups may contain 6 to 12 carbonatoms.

The term “alkylene” as used herein refers to a di-radical alkyl group.Unless otherwise indicated, such groups include saturated hydrocarbonchains containing from 1 to 24 carbon atoms, which may be substituted orunsubstituted, may contain one or more alicyclic groups, and may beheteroatom-containing. “Lower alkylene” refers to alkylene linkagescontaining from 1 to 6 carbon atoms. Examples include, methylene(—CH2-), ethylene (—CH2CH2-), propylene (—CH2CH2CH2-), 2-methylpropylene(—CH2-CH(CH3)-CH2-), hexylene (—(CH2)6-) and the like.

Similarly, the terms “alkenylene,” “alkynylene,” “arylene,”“aralkylene,” and “alkarylene” as used herein refer to di-radicalalkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

The term “amino” is used herein to refer to the group —NRR′ wherein Rand R′ are independently hydrogen or nonhydrogen substituents, withnonhydrogen substituents including, for example, alkyl, aryl, alkenyl,aralkyl, and substituted and/or heteroatom-containing variants thereof.

The terms “halo” and “halogen” are used in the conventional sense torefer to a chloro, bromo, fluoro or iodo substituent.

The term “heteroatom-containing” as in a “heteroatom-containing alkylgroup” (also termed a “heteroalkyl” group) or a “heteroatom-containingaryl group” (also termed a “heteroaryl” group) refers to a molecule,linkage or substituent in which one or more carbon atoms are replacedwith an atom other than carbon, e.g., nitrogen, oxygen, sulfur,phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly,the term “heteroalkyl” refers to an alkyl substituent that isheteroatom-containing, the terms “heterocyclic” or “heterocycle” referto a cyclic substituent that is heteroatom-containing, the terms“heteroaryl” and “heteroaromatic” respectively refer to “aryl” and“aromatic” substituents that are heteroatom-containing, and the like.Examples of heteroalkyl groups include alkoxyaryl,alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like.Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl,pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl,1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containingalicyclic groups are pyrrolidino, morpholino, piperazino, piperidino,tetrahydrofuranyl, etc.

As used herein, the terms “Heterocycle,” “heterocyclic,”“heterocycloalkyl,” and “heterocyclyl” refer to a saturated orunsaturated group having a single ring or multiple condensed rings,including fused bridged and spiroring systems, and having from 3 to 15ring atoms, including 1 to 4 hetero atoms. These ring atoms are selectedfrom the group consisting of nitrogen, sulfur, or oxygen, wherein, infused ring systems, one or more of the rings can be cycloalkyl, aryl, orheteroaryl, provided that the point of attachment is through thenon-aromatic ring. In certain embodiments, the nitrogen and/or sulfuratom(s) of the heterocyclic group are optionally oxidized to provide forthe N-oxide, —S(O)—, or —SO₂— moieties.

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl, and fused heterocycle.

“Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 toabout 30 carbon atoms, including 1 to about 24 carbon atoms, furtherincluding 1 to about 18 carbon atoms, and further including about 1 to12 carbon atoms, including linear, branched, cyclic, saturated andunsaturated species, such as alkyl groups, alkenyl groups, aryl groups,and the like. A hydrocarbyl may be substituted with one or moresubstituent groups. The term “heteroatom-containing hydrocarbyl” refersto hydrocarbyl in which at least one carbon atom is replaced with aheteroatom. Unless otherwise indicated, the term “hydrocarbyl” is to beinterpreted as including substituted and/or heteroatom-containinghydrocarbyl moieties.

By “substituted” as in “substituted hydrocarbyl,” “substituted alkyl,”“substituted aryl,” and the like, as alluded to in some of theaforementioned definitions, is meant that in the hydrocarbyl, alkyl,aryl, or other moiety, at least one hydrogen atom bound to a carbon (orother) atom is replaced with one or more non-hydrogen substituents.Examples of such substituents include, without limitation, functionalgroups, and the hydrocarbyl moieties C1-C24 alkyl (including C1-C18alkyl, further including C1-C12 alkyl, and further including C1-C6alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further includingC2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl(including C2-C18 alkynyl, further including C2-C12 alkynyl, and furtherincluding C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, andfurther including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20aralkyl, and further including C6-C12 aralkyl). The above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated. Unless otherwise indicated, any of the groupsdescribed herein are to be interpreted as including substituted and/orheteroatom-containing moieties, in addition to unsubstituted groups.

By the term “functional groups” is meant chemical groups such as halo,hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl(—CO-alkyl) and C6-C20 arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl),C2-C24 alkoxycarbonyl (—(CO)—O-alkyl), C6-C20 aryloxycarbonyl(—(CO)—O-aryl), halocarbonyl (—CO)—X where X is halo), C2-C24alkylcarbonato (—O—(CO)—O-alkyl), C6-C20 arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH2),mono-substituted C1-C24 alkylcarbamoyl (—(CO)—NH(C1-C24 alkyl)),di-substituted alkylcarbamoyl (—(CO)—N(C1-C24 alkyl)2), mono-substitutedarylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH2), carbamido(—NH—(CO)—NH2), cyano (—C≡N), isocyano (—N₊≡C—), cyanato (—O—C≡N),isocyanato (—O—N₊≡C—), isothiocyanato (—S—C≡N), azido (—N═N+═N—), formyl(—(CO)—H), thioformyl (—(CS)—H), amino (—NH2), mono- and di-(C1-C24alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino,C2-C24 alkylamido (—NH—(CO)-alkyl), C5-C20 arylamido (—NH—(O)-aryl),imino (—CR═NH where R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20alkaryl, C6-C20 aralkyl, etc.), alkylimino (—CR═N(alkyl), whereR=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (—CR═N(aryl), whereR=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO2), nitroso (—NO),sulfo (—SO2-OH), sulfonato (—SO2-O—), C1-C24 alkylsulfanyl (—S-alkyl;also termed “alkylthio”), arylsulfanyl (—S-aryl; also termed“arylthio”), C1-C24 alkylsulfinyl (—(SO)-alkyl), C5-C20 arylsulfinyl(—(SO)-aryl), C1-C24 alkylsulfonyl (—SO2-alkyl), C5-C20 arylsulfonyl(—SO2-aryl), phosphono (—P(O)(OH)2), phosphonato (—P(O)(O-)2),phosphinato (—P(O)(O—)), phospho (—PO2), and phosphino (—PH2), mono- anddi-(C1-C24 alkyl)-substituted phosphino, mono- and di-(C5-C20aryl)-substituted phosphine. In addition, the aforementioned functionalgroups may, if a particular group permits, be further substituted withone or more additional functional groups or with one or more hydrocarbylmoieties such as those specifically enumerated above.

By “linking” or “linker” as in “linking group,” “linker moiety,” etc.,is meant a bivalent radical moiety that connects two groups via covalentbonds. Examples of such linking groups include alkylene, alkenylene,alkynylene, arylene, alkarylene, aralkylene, and linking moietiescontaining functional groups including, without limitation: amido(—NH—CO—), ureylene (—NH—CO—NH—), imide (—CO—NH—CO—), epoxy (—O—),epithio (—S—), epidioxy (—O—O—), carbonyldioxy (—O—CO—O—), alkyldioxy(—O—(CH2)n-O—), epoxyimino (—O—NH—), epimino (—NH—), carbonyl (—CO—),etc. Any convenient orientation and/or connections of the linkers to thelinked groups may be used.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl and aryl” isto be interpreted as “substituted alkyl and substituted aryl.”

In certain embodiments, a substituent may contribute to opticalisomerism and/or stereo isomerism of a compound. Salts, solvates,hydrates, and prodrug forms of a compound are also of interest. All suchforms are embraced by the present disclosure. Thus the compoundsdescribed herein include salts, solvates, hydrates, prodrug and isomerforms thereof, including the pharmaceutically acceptable salts,solvates, hydrates, prodrugs and isomers thereof. In certainembodiments, a compound may be a metabolized into a pharmaceuticallyactive derivative.

Unless otherwise specified, reference to an atom is meant to includeisotopes of that atom. For example, reference to H is meant to include1H, 2H (i.e., D) and 3H (i.e., T), and reference to C is meant toinclude 12C and all isotopes of carbon (such as 13C).

Definitions of other terms and concepts appear throughout the detaileddescription below.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As summarized above, compounds and methods are provided for thetreatment of pathogen infections, where the compound inhibits an enzymein the phosphatidylinositol 4,5-bisphosphate (PIP-2) synthetic pathway,including without limitation selective inhibition of PI4-kinase. In someembodiments, the anti-infective compounds have broad spectrum activityagainst a variety of infective diseases, where the diseases are causedby pathogens containing a basic amino acid PIP-2 pincer (BAAPP) domainthat interacts with phosphatidylinositol 4,5-bisphosphate (PIP-2) tomediate replication.

In some embodiments, an anti-infective compound that is a PI4-kinaseinhibiting compound is contacted with a pathogen, in a dose and for aperiod of time sufficient to inhibit replication. Contacting may beperformed in vitro or in vivo. Such PI4-kinase inhibiting compounds mayinhibit pathogen replication by inhibiting the production of PIP-2.

In some embodiments a method of inhibiting a PI4-kinase, including butnot limited to a class III PI4-kinase, are provided, where a compound ofthe invention is brought into contact with a PI4-kinase in a dose andfor a period of time sufficient to inhibit activity of the enzyme.

Also provided are pharmaceutical compositions that include the subjectcompounds, where a compound of the invention is formulated with apharmaceutically acceptable excipient. Formulations may be provided in aunit dose, where the dose provides an amount of the compound effectiveto achieve a desired result, including without limitation inhibition ofpathogen replication.

These compounds and methods find use in a variety of applications inwhich inhibition of a PI-kinase is desired.

Compounds

As summarized above, aspects of the disclosure include PI4-kinaseinhibitor compounds. In some cases, the compounds include a5-aryl-thiazole core structure. The aryl ring may be a 6-memberedheteroaryl or phenyl ring that includes a further substituent meta tothe thiazole ring substituent. The thiazole ring of the core structuremay include further substituents at the 2- and/or 4-positions of thering. In some embodiments, the PI4-kinase inhibitor compounds are2-amino-5-phenylthiazole compounds that include a thiazole ring havingan amino substituent at the 2-position of the ring, and a phenylsubstituent at the 5-position of the ring. In some embodiments, thecompound includes further substituents, such as a substituent at eitherthe 4 or 5-position of the thiazole ring. The aryl ring of the corestructure may be further substituted with any convenient substituentsincluding but not limited to alkyl, acyloxy, aminoalkoxy, cyano,halogen, hydroxyl, nitro, —NHCOR, —SO₂NHR, —CONHR or —NHSO₂R, where R isalkyl, heteroalkyl, heterocycle or aryl. Exemplary 5-aryl-thiazolecompounds are set forth in the following structures and formulaeI-XVIII.

In some cases, the subject compound is described by the structure offormula (I):

where:

-   -   Z¹ and W are each independently a covalent bond or a linking        functional group;    -   Y¹ and Y² are each independently CR² or N;    -   R¹ is selected from hydrogen, an alkyl, an aryl, an        alkyl-heterocycle and a heterocycle;    -   R³ selected from hydrogen and an alkyl;    -   R⁴ is selected from an alkyl, an aralkyl, an aryl, an        alkyl-cycloalkyl, a cycloalkyl, an alkyl-heterocycle, a        heterocycle, an amino or an alkoxy; and    -   R², R⁶ and R⁷ are independently selected from hydrogen, an        alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle,        a cyano, a halogen, an amino, an acyl, an acyloxy, an amido, and        a nitro.

In certain embodiments, in formula (I), R¹ to R⁴ are independentlyselected from corresponding groups as depicted in any of the structuresof Table 1.

In some embodiments, in formula (I), Y¹ is CH and Y² is CR², such thatthe compound is described by the structure:

where:

-   -   Z¹ and W are each independently a covalent bond or a linking        functional group;    -   R¹ is selected from an alkyl, an aryl, an alkyl-heterocycle and        a heterocycle;    -   R² is selected from hydrogen, a halogen, an alkyl and an alkoxy;    -   R³ is selected from hydrogen and an alkyl;    -   R⁴ is selected from an alkyl, an aralkyl, an aryl, an        alkyl-cycloalkyl, a cycloalkyl, an alkyl-heterocycle, a        heterocycle; and    -   R⁶ and R⁷ are independently selected from hydrogen, an alkyl, an        aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano,        a halogen, an amino, an acyl, an acyloxy, an amido and nitro.

In certain embodiments, R² is selected from hydrogen, a halogen and analkoxy.

In some embodiments, R³ and R⁶ are selected such that they form a6-membered ring as part of a fused tricyclic aryl-thiazole corestructure.

In some embodiments, R¹ is not a hydroxy-substituted alkyl group, suchas —(CH₂)₂—OH.

In some embodiments, R¹ is selected from hydrogen, an alkyl, an aryl(e.g., a phenyl), an alkyl-heterocycle and a heterocycle (e.g., pyridyl,pyrimidinyl, pyrrolyl, pyrrolidinyl, quinolinyl, indolyl, furyl,imidazolyl, oxazolyl, thiazolyl, 1,2,4-triazolyl, tetrazolyl,pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl). Insome embodiments, R¹ is selected from hydrogen, a substituted loweralkyl (e.g., a substituted methyl or ethyl), a phenyl, a cycloalkyl, apyridyl and a pyrimidinyl.

In some instances, R⁴ is —(CH₂)_(n)—R¹⁰, where n is 0, 1, 2 or 3; andR¹⁰ is a cycloalkyl or a heterocycle (e.g., a 5- or 6-membered saturatedN-containing heterocycle). In certain cases, R¹⁰ is selected from acyclohexyl, a cyclopentyl, a cyclopropyl, a lower alkyl, a pyrrolidinyland a piperidinyl.

In some embodiments, the subject compound is described by the structureof formula (II):

where:

-   -   Z¹ and Z² are each independently a covalent bond or a linking        functional group;    -   R¹ is selected from hydrogen, an alkyl (e.g., a substituted        ethyl, or a heterocycle-substituted lower alkyl), an aryl (e.g.,        a phenyl), and a heterocycle (e.g., a pyridyl, a pyrimidinyl);    -   R² is selected from hydrogen, a halogen and an alkoxy;    -   R³ and R⁵ are selected from hydrogen and an alkyl (e.g., a lower        alkyl such as a methyl);    -   R⁴ is selected from an alkyl (e.g., a cycloalkyl such as        cycloheptyl, cyclohexyl, cyclopentyl, cyclopropyl or a lower        alkyl such as methyl, ethyl or tert-butyl), an aralkyl (e.g., a        benzyl or a phenylethyl), an aryl, an alkyl-heterocycle, a        heterocycle, an amino and an alkoxy; and    -   R⁶ and R⁷ are independently selected from hydrogen, an alkyl, an        aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano,        a halogen (e.g., fluoro, chloro or bromo), an amino, an acyl, an        acyloxy, an amido, and a nitro.

In certain embodiments, in formula (II), R¹ to R⁴ are independentlyselected from corresponding groups as depicted in any of the structuresof Table 1.

The linking functional group may be any convenient bivalent group.Linking functional groups of interest include, but are not limited to,an amino, an amido, an ester, a carbonyloxy, an ether, a carbamate, asulfonamide, a carbonyl, a sulfonyl, a sulfinyl, or the like. In someembodiments, the linking functional group is described by one of thefollowing formulas: —SO₂NR—, —NR—, —NRC(═O)—, or —NRC(═O)NR— where eachR is independently H, an alkyl, a cycloalkyl, a heterocycle, aheterocycloalkyl, an aryl or a heteroaryl; —O—; —C(═O) —; —C(═O)X— whereX is NR, O or S and where R is H or an alkyl; —S(═O)— or —SO₂—; wherefor each of the formulae depicted it is understood that both possibleorientations of a functional group are included. In some embodiments, informula (I), Z¹ is —SO₂NH— or —CONH— and W is a covalent bond, —NR— or—NRC(═O)—, where R is H or an alkyl. In some embodiments, in formula(II), Z¹ is —NHSO₂— or —SO₂NH—; and Z² is a covalent bond or —C(═O)—.

In some embodiments, R¹ is described by the formula—(CH₂)_(n)—CH(R⁸)—CHR⁹, where R⁸ is hydrogen or a lower alkyl (e.g.,methyl) and R⁹ is hydrogen, an aryl (e.g., a phenyl) or a heterocycle(e.g., pyridyl (e.g., 3-pyridyl), pyrimidinyl, pyrrolyl, pyrrolidinyl,quinolinyl, indolyl, furyl, imidazolyl, oxazolyl, thiazolyl,1,2,4-triazolyl, tetrazolyl, pyrrolidino, morpholino, piperazino,piperidino, tetrahydrofuranyl); and n is 0, 1, 2 or 3. In someembodiments, n is 0. In certain embodiments, R¹ is a substituted ethylgroup, for example, a group described by one of the followingstructures:

In other embodiments, R¹ is described by the formula:

-   -   where A is a 6-membered aryl, heteroaryl, heterocyclyl, or        cycloalkyl, where Z¹¹-Z¹⁶ are independently selected from N,        CR′, NR and CR′R″, where R is H or alkyl, and R′ and R″ are        independently selected from hydrogen, an alkyl, an aryl, a        hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a        halogen (e.g., fluoro, chloro or bromo), an amino, an acyl, an        acyloxy, an amido, and a nitro.

In some embodiments, R¹ is described by the following formula:

-   -   where Z¹³ is CR²³ or N, where R²²-R²⁶ are independently selected        from hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy, an        aryloxy, a heterocycle, a cyano, a halogen (e.g., fluoro, chloro        or bromo), an amino, an acyl, an acyloxy, an amido, and a nitro.

In certain embodiments, R¹ is described by the following formula:

-   -   where Z¹³ is CR²³ or N, where R²³, R²⁴ and R²⁶ are independently        selected from hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy,        an aryloxy, a heterocycle, a cyano, a halogen (e.g., fluoro,        chloro or bromo), an amino, an acyl, an acyloxy, an amido, and a        nitro.

In some embodiments, R¹ is described by the following formula:

where Z³ is N or CR¹¹; Z⁴ is N or CR¹³; and R¹¹ to R¹⁵ are eachindependently selected from where R²²-R²⁶ are independently selectedfrom hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, aheterocycle, a cyano, a halogen (e.g., fluoro, chloro or bromo), anamino, an acyl, an acyloxy, an amido, and a nitro. In certainembodiments, R¹¹ to R¹⁵ are each independently selected from hydrogen,an alkyl, an alkoxy, an acyloxy, a cyano, a halogen, and hydroxyl. Incertain embodiments, Z³ is CR¹¹, Z⁴ is CR¹³; R¹¹, R¹⁴ and R¹⁵ are eachhydrogen; R¹² is hydrogen, an alkoxy (e.g., methoxy) or a halogen (e.g.,fluoro); and R¹³ is selected from hydrogen, acetyloxy, hydroxy, methoxy,cyano-methyl and halogen (e.g., fluoro). In certain embodiments, Z⁴ isN. In certain embodiments, Z³ and Z⁴ are each N.

In some instances, R¹ is described by the following formula:

-   -   where Z¹³ and Z¹⁴ are each independently CR′R″ or NR, where R is        H or alkyl, and R³², R³⁵, R³⁶, R′ and R″ are independently        selected from hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy,        an aryloxy, a heterocycle, a cyano, a halogen (e.g., fluoro,        chloro or bromo), an amino, an acyl, an acyloxy, an amido, and a        nitro. In certain embodiments, R³², R³⁵, R³⁶, R′ and R″ are each        independently selected from hydrogen, an alkyl, an alkoxy, an        acyloxy, a cyano, a halogen, and hydroxyl.

In certain embodiments, R¹ is described by one of the followingformulas:

In some embodiments, in formulae (I) or (II), R² is methoxy. In someembodiments, in formulae (I) or (II), R³ is methyl.

In some embodiments, in formula (II), Z² is a covalent bond or —C(═O)—,and R⁴ is a lower alkyl (trifluoromethyl, tert-butyl, methyl, ethyl), acycloalkyl (e.g., cyclopentyl, 1-fluoro-cyclopentyl or cyclohexyl) or—CH₂-cycloalkyl, a heterocycle (e.g., a N-linked saturated heterocyclesuch as N-pyrollidinyl, N-morpholino), or an amino (e.g., an amino-alkylsuch as N-amino-cyclopentyl).

In some embodiments, in formula (II), R⁴ is described by the formula—NR¹⁶R¹⁷, wherein R¹⁶ and R¹⁷ are each independently selected fromhydrogen, an alkyl, a cycloalkyl, and wherein optionally R¹⁶ and R¹⁷ arecyclically linked (e.g., to form a N-heterocyclyl).

In some embodiments, in formula (II), Z² is a covalent bond; and R⁴ isan alkyl or an alkyl-cycloalkyl (e.g., 1-cyclopentyl-methyl-).

In some embodiments, in formula (II), R⁴ is selected from methyl,trifluoromethyl, ethyl, tert-butyl, cyclopentyl, N-pyrrolidinyl,N-morpholinyl, N-amino-cyclopentyl and 1-fluoro-cyclopentyl.

In certain embodiments, in formula (II), R⁵ is hydrogen. In certainembodiments, in formula (II), R⁶ and R⁷ are each hydrogen.

In some instances, the compound is described by the structure of formula(III):

-   -   where R¹ is selected from hydrogen, an alkyl, an aryl, an        alkyl-heterocycle and a heterocycle;    -   R² is hydrogen, alkyl or alkoxy;    -   R³ is alkyl;    -   R⁵ is H or alkyl;    -   R⁴ is lower alkyl, cycloalkyl, -alkyl-cycloalkyl, heterocyclyl        or alkyl-heterocyclyl (e.g., —(CH₂)_(n)-cycloalkyl or        —(CH₂)_(n)-heterocycyl, where n is 0, 1 or 2);    -   W¹ is —SO₂— or —C(═O)—; and    -   W² is a covalent bond, —NH—, or —NHCO—.

In certain embodiments, in formula (III), R¹ is described by thefollowing structure:

-   -   where A is a 6-membered aryl, heteroaryl, heterocyclyl, or        cycloalkyl, where Z¹¹-Z¹⁶ are independently selected from N,        CR′, NR and CR′R″, where R is H or alkyl, and R′ and R″ are        independently selected from hydrogen, an alkyl, an aryl, a        hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a        halogen (e.g., fluoro, chloro or bromo), an amino, an acyl, an        acyloxy, an amido, and a nitro.

In some embodiments, in formula (III), R¹ is described by one of thefollowing structures:

-   -   where Z¹³ is CR²³ or N, where Z³ is N or CR¹¹; Z⁴ is N or CR¹³;        and R¹¹ to R¹⁵ and R²³-R²⁶ are each independently selected from        hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, a        heterocycle, a cyano, a halogen (e.g., fluoro, chloro or bromo),        an amino, an acyl, an acyloxy, an amido, and a nitro.

In certain embodiments, in formula (III), R¹ is described by thefollowing:

-   -   where Z¹³ is CR²³ or N, and R²³, R²⁴ and R²⁶ are independently        selected from hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy,        an aryloxy, a heterocycle, a cyano, a halogen (e.g., fluoro,        chloro or bromo), an amino, an acyl, an acyloxy, an amido, and a        nitro. In certain embodiments, R²³, R²⁴ and R²⁶ are        independently selected from H, alkyl (e.g., methyl or ethyl),        alkoxy (e.g., methoxy or ethoxy) and halo (e.g., fluoro or        chloro). In certain cases, Z¹³ is selected from CH and N.

In certain embodiments, in formula (III), R¹ to R⁴ are independentlyselected from corresponding groups as depicted in any of the structuresof Table 1.

In some cases, the compound is described by the structure of formula(IV):

-   -   where:    -   R¹ is —(CH₂)_(n)—R²⁰, where R²⁰ is an aryl, a cycloalkyl or a        heterocycle and n is 0, 1 or 2;    -   R² is H, alkyl or alkoxy;    -   R³ is H or alkyl;    -   W² is a covalent bond, —NH—, or —NHCO—;    -   n is 0, 1, 2 or 3; and    -   R¹⁰ is a cycloalkyl or a heterocycle.

In certain cases, in formula (IV), R¹ is a phenyl, a pyridyl, adiazinyl, a piperidinyl, a piperazinyl, or a pyrriloidinyl.

In certain cases, in formula (IV), R¹ is described by the followingstructure:

-   -   where A is a 6-membered aryl, heteroaryl, heterocyclyl, or        cycloalkyl, where Z¹¹-Z¹⁶ are independently selected from N,        CR′, NR and CR′R″, where R is H or alkyl, and R′ and R″ are        independently selected from hydrogen, an alkyl, an aryl, a        hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a        halogen (e.g., fluoro, chloro or bromo), an amino, an acyl, an        acyloxy, an amido, and a nitro.

In some cases, the compound is described by the structure of formula(V):

-   -   where: R² is alkoxy, R³ is lower alkyl, W² is a covalent bond,        —NH—, or —NHCO—; n is 0, 1 or 2; Z¹³ is N or CR²³, R¹⁰ is a        cycloalkyl or a heterocycle; and R²³-R²⁶ are independently        selected from hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy,        an aryloxy, a heterocycle, a cyano, a halogen (e.g., fluoro,        chloro or bromo), an amino, an acyl, an acyloxy, an amido, and a        nitro. In certain embodiments, R²³-R²⁶ are independently        selected from hydrogen, halo, alkyl, and alkoxy.

In some instances, the compound is described by the structure of one offormulae (VI), (VII) or (VIII):

-   -   where: R² is alkoxy, R³ is lower alkyl, n is 0, 1 or 2; Z¹³ is N        or CR²³, R¹⁰ is a cycloalkyl or a heterocycle; and R²³-R²⁶ are        independently selected from hydrogen, an alkyl, an aryl, a        hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a        halogen (e.g., fluoro, chloro or bromo), an amino, an acyl, an        acyloxy, an amido, and a nitro. In certain embodiments, R²³-R²⁶        are independently selected from hydrogen, halo, alkyl, and        alkoxy.

In certain instances, R¹⁰ is a cyclopentyl, a cyclohexyl, a piperidinylor a pyrrolidinyl. In certain embodiments, in formulae (IV)-(VIII), R²is methoxy. In certain embodiments, in formulae (IV)-(VIII), R³ ismethyl.

In certain embodiments, in formulae (IV)-(VIII), Z¹³, R²³-R²⁶, R², R³and R¹⁰ are independently selected from corresponding groups as depictedin any of the structures of Table 1.

In some cases, the compound is described by the structure of formula(IX):

-   -   where: R² is alkoxy; R³ is lower alkyl; W² is a covalent bond,        —NH—, or —NHCO—; each n is independently 0, 1 or 2; Z¹³ is N or        CR²³; R¹⁰ is a cycloalkyl or a heterocycle; and R²⁰ is an aryl,        a cycloalkyl or a heterocycle.

In certain cases, R²⁰ and R¹⁰ are independently described by thefollowing structure:

-   -   where A is a 6-membered aryl, heteroaryl, heterocyclyl, or        cycloalkyl, where Z¹¹-Z¹⁶ are independently selected from N,        CR′, NR and CR′R″, where R is H, an alkyl, a cycloalkyl, a        heterocycloalkyl, an aryl or a heteroaryl; and R′ and R″ are        independently selected from hydrogen, an alkyl, an aryl, a        hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a        halogen (e.g., fluoro, chloro or bromo), an amino, an acyl, an        acyloxy, an amido, and a nitro.

In certain embodiments, R²⁰ is a phenyl, a pyridyl, a diazinyl, apiperidinyl, a piperazinyl, or a pyrrolidinyl.

In some instances, the compound is described by the structure of one offormulae (X), (XI) or (XII):

-   -   where R² is an alkoxy, R³ is a lower alkyl, each n is        independently 0, 1 or 2; R¹⁰ is a cycloalkyl or a heterocycle;        and R²⁰ is an aryl, a cycloalkyl or a heterocycle. In certain        embodiments, R²⁰ is a phenyl, a pyridyl, a diazinyl, a        piperidinyl, a piperazinyl, or a pyrrolidinyl.

In certain instances, R¹⁰ is a cyclopentyl, a cyclohexyl, a piperidinylor a pyrrolidinyl. In certain instances, R²⁰ is a phenyl, or a pyridyl.In certain embodiments, in formulae (IX)—(XII), R² is methoxy. Incertain embodiments, in formulae (IX)—(XII), R³ is methyl.

In some embodiments, the compound is described by the structure offormula (XIII):

-   -   where R¹ is an alkyl, an aryl, an alkyl-heterocycle or a        heterocycle; and R⁴ is an alkyl, an aralkyl, an aryl, an        alkyl-cycloalkyl, a cycloalkyl, an alkyl-heterocycle, or a        heterocycle. In certain instances, R⁴ is a cyclopentyl, a        cyclohexyl, a piperidinyl or a pyrrolidinyl. In certain        instances, R¹ is a phenyl or a pyridiyl.

In certain embodiments, in formulae (I)-(XIII), R¹ or R²⁰ is describedby one of the following structures:

-   -   where R⁴⁴-R⁴⁶ are independently selected from hydrogen, an        alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle,        a cyano, a halogen (e.g., fluoro, chloro or bromo), an amino, an        acyl, an acyloxy, an amido, and a nitro; R′ is hydrogen, an        alkyl, an aryl or a heterocycle; n² is 0, 1, 2 or 3, and n¹ is        0, 1 or 2; and    -   R⁴ is —(CH₂)_(n)-cycloalkyl (e.g., cyclopropyl, cyclopentyl or        cyclohexyl), —(CH₂)_(n)-heterocycle (e.g., piperidinyl, a        piperazinyl, or a pyrriloidinyl), or lower alkyl, where each n        is independently 0, 1, 2 or 3. In certain embodiments, in        formula (XIII), n is 1.

In certain embodiments, in formula (XIII), R¹ and R⁴ are independentlyselected from corresponding groups as depicted in any of the structuresof Table 1.

In certain embodiments, the compound is described by one of thefollowing structures:

-   -   where R¹ is selected from a phenyl, a pyridyl, a diazinyl, a        piperidinyl, a piperazinyl, a pyrriloidinyl and —(CH₂)_(n)—R²⁰        where R²⁰ is an aryl, a cycloalkyl or a heterocycle and n is 0,        1 or 2.

In certain embodiments, in the nine structures depicted above, R¹ isdescribed by one of the following structures:

-   -   where R⁴⁴-R⁴⁶ are independently selected from hydrogen, an        alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle,        a cyano, a halogen (e.g., fluoro, chloro or bromo), an amino, an        acyl, an acyloxy, an amido, and a nitro; R′ is hydrogen, an        alkyl, an aryl or a heterocycle; n² is 0, 1, 2 or 3, and n¹ is        0, 1 or 2. In certain embodiments, R⁴⁴-R⁴⁶ are independently        selected from H, an alkyl, an alkoxy, hydroxyl, and a halo        (e.g., fluoro or chloro).

In some embodiments, the compound is described by the structure offormula (XIV):

-   -   where R¹ is a phenyl, a pyridyl (e.g., 4-pyridyl or 3-pyridyl)        or a pyrimidinyl (e.g., a 4-pyrimidinyl or 3-pyrimidinyl).

In some embodiments, in formula (XIV), R⁵-R⁷ are each hydrogen.

In certain embodiments, in formula (XIV), R¹ to R⁵ are independentlyselected from corresponding groups as depicted in any of the structuresof Table 1.

In some embodiments, in formula (XIV), R¹ is a phenyl, and R⁵-R⁷ areeach hydrogen. In certain embodiments, the compound is described by thestructure of formula (XV):

-   -   where Z³ is N or CR¹¹; Z⁴ is N or CR¹³; and R¹¹-R¹⁵ are each        independently selected from hydrogen, an alkyl (e.g., a lower        alkyl such as methyl or trifluoromethyl), an aryl, a hydroxy, an        alkoxy, an aryloxy, a heterocycle, a cyano, a halogen (e.g.,        fluoro, chloro or bromo), an amino (e.g., —NMe₂), an acyl, an        acyloxy, an amido, or a nitro.

In some embodiments, in formula (XV), R² is an alkoxy (e.g., methoxy).In some embodiments, in formula (XV), R³ is an alkyl (e.g., methyl).

In some embodiments, in formula (XV), Z⁴ is CR¹³ and Z³ is CR¹¹. In someembodiments, in formula (XV), Z⁴ is N and Z³ is CR¹¹. In someembodiments, in formula (XV), Z³ and Z⁴ are each N.

In some embodiments, in formula (XV), R¹¹-R¹⁵ are each independentlyselected from hydrogen, an alkoxy (e.g., methoxy), a halogen (e.g.,fluoro), acyloxy (e.g., acetyloxy), hydroxy and cyano-alkyl (e.g.,cyano-methyl).

In some embodiments, the subject compound is described by the structureof formula (XVI):

-   -   where R¹⁷ is hydrogen, an alkoxy (e.g., methoxy) or a halogen        (e.g., fluoro); and R¹⁸ is selected from hydrogen, acetyloxy,        hydroxy, methoxy, cyano-methyl and halogen (e.g., fluoro).

In some embodiments, in formula (XVI), R⁴ is selected from methyl,trifluoromethyl, ethyl, tert-butyl, cyclopentyl, N-pyrrolidinyl,N-morpholinyl, N-amino-cyclopentyl and 1-fluoro-cyclopentyl; and R¹⁷ andR¹⁸ are independently selected from hydrogen, methoxy, fluoro,acetyloxy, hydroxy, and cyano-methyl.

In some embodiments, the subject compound is described by the structureof formula (XVII):

-   -   wherein:    -   Z¹ is —NHSO₂— or —SO₂NH—;    -   Z² is a covalent bond or —C(═O)—;    -   Z³ is N or CR¹¹;    -   Z⁴ is N or CR¹³; and    -   R¹¹ to R¹⁵ are each independently selected from hydrogen, an        alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle,        a cyano, a halogen (e.g., fluoro, chloro or bromo), an amino, an        acyl, an acyloxy, an amido, and a nitro. In certain embodiments,        in formula (XVII), R¹¹ to R¹⁵ are each independently selected        from hydrogen, an alkoxy, an acyloxy, a halogen, and hydroxyl.

In some embodiments, in formula (XVII), Z³ is CR¹¹, Z⁴ is CR¹³; R¹¹,R¹², R¹⁴ and R¹⁵ are each hydrogen; and R¹³ is selected from hydrogen,acetyloxy, hydroxy, methoxy and halogen (e.g., fluoro).

In some embodiments, in formula (XVII), Z³ is CR¹¹, Z⁴ is N, and R¹¹,R¹², R¹⁴ and R¹⁵ are each hydrogen.

In some embodiments, in formula (XVII), Z³ and Z⁴ are each N, and R¹²,R¹⁴ and R¹⁵ are each hydrogen.

In some embodiments, in formula (XVII), Z² is a covalent bond or—C(═O)—, and R⁴ is a lower alkyl (e.g., trifluoromethyl, tert-butyl), acycloalkyl (e.g., cyclopentyl or 1-fluoro-cyclopentyl) or—CH₂-cycloalkyl (e.g., —CH₂-cyclopentyl), a heterocycle (e.g., aN-linked saturated heterocycle such as N-pyrrolidino or N-morpholino),or an amino (e.g., an amino-alkyl such as N-amino-cyclopentyl).

In some embodiments, in formula (XVII), Z² is —C(═O)—, and R⁴ isdescribed by the formula —NR¹⁶R¹⁷, where R¹⁶ and R¹⁷ are eachindependently selected from hydrogen, an alkyl, and a cycloalkyl,wherein optionally R¹⁶ and R¹⁷ are cyclically linked (e.g., to form aN-heterocyclyl).

In some embodiments, in formula (XVII), Z² is a single bond; and R⁴ isan alkyl or a cycloalkyl-alkyl (e.g., 1-cyclopentyl-methyl).

In some embodiments, in formula (XVII), Z² is —C(═O)—, and R⁴ isselected from trifluoromethyl, ethyl, tert-butyl, N-pyrrolidinyl,N-morpholinyl, N-amino-cyclopentyl and 1-fluoro-cyclopentyl.

In some embodiments, the subject compound is described by the structureof formula (XVIII):

-   -   where X and Y are independently selected from the substituents        groups shown below:

In certain embodiments, in formula (XVIII), R¹ and R⁴ are independentlyselected from corresponding groups as depicted in any of the structuresof Table 1.

In some embodiments, the subject compound is described by the structureof compound PT423, shown in Table 1. In some embodiments, the subjectcompound is described by one of the structures labeled M1, M2, and M3 inFIG. 2.

In certain embodiments, the compound is described by the structure ofone of the compounds of Table 1.

TABLE 1 Compounds Cmpd Structure #

AB3454

AB3455

AB3456

AB3457

AB3458

AB3459

AB3460

AB3462

AB3463

AB3464

AB3480

AB3620

AB3621

AB3622

AB3624

AB3625

AB3660

AB3661

AB3662

AB3663

AB3664

AB3665

AB3666

AB3667

AB3668

AB3669

AB3670

AB3671

AB3672

AB3673

AB3674

AB3675

AB3676

AB3677

AB3678

AB3680

AB3681

AB3682

AB3725

AB3726

AB3727

AB3728

AB3729

AB3730

AB3731

AB3732

AB3733

AB3734

AB3735

AB3736

AB3737

AB3738

AB3742

AB3748

AB3749

AB3750

AB3751

AB3752

AB3753

AB3754

AB3755

AB3756

AB3757

AB3758

AB3759

AB3760

AB3761

AB3762

AB3763

AB3764

AB3765

AB3766

AB3767

AB3768

AB3769

AB3770

AB3786

AB3787

AB3788

AB3789

AB3797

AB3798

AB3800

AB3801

AB3802

AB3803

AB3805

AB3806

AB3807

AB3808

AB3809

AB3811

AB3823

AB3824

AB3825

AB3826

AB3827

AB3828

AB3829

AB3830

AB3831

AB3832

AB3833

AB3834

AB3835

AB3836

AB3837

AB3855

AB3856

AB3858

AB3859

AB3860

AB3861

AB3862

AB3863

AB3864

AB3865

AB3866

AB3867

AB3868

AB3869

AB3886

AB3913

AB3914

AB3915

AB3916

AB3917

AB3918

AB3919

AB3920

AB3921

AB3922

AB3923

AB3924

AB3925

AB3926

AB3943

AB3944

AB3945

AB3946

AB3947

AB3948

AB3949

AB3950

AB3951

AB3952

AB3953

AB3954

AB3955

AB3956

AB3957

AB3958

AB3977

AB3978

AB3979

AB3980

AB3981

AB4021

AB4022

AB4023

AB4024

AB4025

AB4050

AB4051

AB4052

AB4053

AB4054

AB4094

AB4095

AB4096

AB4097

AB4098

AB4099

AB4100

AB4106

AB4107

AB4108

AB4109

AB4110

AB4111

AB4112

AB4113

AB4114

AB4115

AB4116

AB4117

AB4118

AB4119

AB4137

AB4138

AB4139

AB4140

AB4141

AB4142

AB4143

AB4144

AB4145

AB4146

AB4147

AB4148

AB4149

AB4150

AB4162

AB4163

AB4164

AB4165

AB4166

AB4167

AB4168

AB4169

AB4170

AB4171

AB4172

AB4173

AB4174

AB4175

AB4176

AB4177

AB4178

AB4179

AB4180

AB4181

AB4182

AB4183

AB4184

AB4185

AB4186

AB4188

AB4189

AB4190

AB4191

AB4192

AB4193

AB4207

AB4208

AB4225

AB4226

AB4227

AB4228

AB4229

AB4230

AB4231

AB4232

AB4233

AB4234

AB4235

AB4236

AB4237

AB4238

AB4239

AB4240

AB4241

AB4242

AB4243

AB4244

PT41

PT42

PT43

PT44

PT45

PT46

PT47

PT48

PT49

PT410

PT411

PT412

PT413

PT414

PT415

PT416

PT417

PT418

PT419

PT420

PT421

PT422

PT423

PT424

PT425

Optimization for Bioavailability and Metabolic Stability

In some embodiments, the subject compounds are provided by oral dosingand absorbed into the bloodstream. In some embodiments, the oralbioavailability of the subject compounds is 30% or more. Modificationsmay be made to the subject compounds or their formulations using anyconvenient methods to increase absorption across the gut lumen or theirbioavailability.

In some embodiments, the subject compounds are metabolized in vivo toproduce one or more metabolites. In some embodiments, the subjectcompounds may be optimized for metabolic stability using any convenientmethods. In some embodiments, the subject compounds are metabolicallystable (e.g., remain substantially intact in vivo during the half-lifeof the compound). In certain embodiments, the compounds have a half-life(e.g., an in vivo half-life) of 5 minutes or more, such as 10 minutes ormore, 12 minutes or more, 15 minutes or more, 20 minutes or more, 30minutes or more, 60 minutes or more, 2 hours or more, 6 hours or more,12 hours or more, 24 hours or more, or even more.

In some embodiments, one or more metabolites exhibits similar or greateractivity against the relevant target kinase(s), or against a particularpathogen, than does the parent compound. In some embodiments, thesubject compounds are metabolized by a hydroxylation, a deacylation or adearylation process that would also provide a direct route to type IIconjugation and excretion of the compound. Modifications may be made tothe subject compounds using any convenient methods to deactivate oractivate these metabolic processes. For example, analogs of the compoundPT423 may be prepared with modifications to the acylamide moiety whichmay decrease accessibility to nucleophilic attack, decrease lability tohydrolysis by changing the linkage to the more stable urea, and ablatesites of hydroxylation by replacing the modified carbon by nitrogen orby derivatization with fluoride. In some embodiments, modifications aremade to a subject compound to be consistent with structure-activitydata, so that the modifications are tolerated with respect to PI4-kinase inhibiting activity.

In some cases, a N-dearylation process may contribute to metabolism ofthe subject compounds. To modulate this dearylation process, analogs ofPT423 are synthesized with modifications to the N-phenylsulfonamidemoiety, such as reversing a sulfonamide bond, altering the electronicsof the phenyl ring by addition of electron-withdrawing or donatingsubstituents, hindering the formation of a non-aromatic intermediate byeither adding a carbon bond to oxygen, or by replacing the phenyl ringwith heterocycles (e.g. pyrimidinyl, 3- or 4-pyridinyl, etc).

PI-Kinase Inhibition

As summarized above, aspects of the invention include PI4-kinaseinhibiting compounds, and methods of inhibition using the same. ThePI4-kinase inhibiting compounds are compounds that inhibit the activityof a PI4-kinase in a cell, upon contact with a cell or componentsthereof.

In some instances, the types of cells in which the compounds of theinvention exhibit activity are ones that have been infected with apathogen, as described herein.

By inhibiting a PI-kinase it is meant that the activity of the enzyme isdecreased by a factor of 2 or more, such as 3 or more, 5 or more, 10 ormore, 100 or more, or 1000 or more, relative to its normal activity(e.g., relative to a positive control).

In some embodiments, the subject compounds are inhibitors of aPI3-kinase. In some embodiments, the subject compounds are inhibitors ofa PI4-kinase, such as a PI4-III-kinase (e.g., PI4-IIIα or PI4-IIIβ). Insome embodiments, the subject compounds have a PI-kinase inhibitionprofile that reflects activity against two or more PI-kinases. In someembodiments, the subject compounds specifically inhibit both a type IIPI3-kinase, such as PI3-kinase IIβ, and a type III PI4-kinase, such asPI4K-IIIα and/or PI4K-IIIβ). In some embodiments, the subject compoundsspecifically inhibit a PI4-kinase without undesired inhibition ofprotein kinases. In some embodiments, the subject compounds specificallyinhibit a PI4-kinase without undesired inhibition of PI3-kinase. In someembodiments, the subject compounds specifically inhibit a PI4-kinaseand/or a specific PI3-kinase subclass without undesired inhibition ofother PI3-kinase subclasses or protein kinases.

In some embodiments, the compounds of the disclosure interfere with theinteraction of a BAAPP domain with PIP2 in a pathogen (e.g., HCV). Forexample, the subject compounds may act by decreasing the levels of PIP2either directly or indirectly that bind specifically to the BAAPP domainof the pathogen. In general, pathogens that include a BAAPP domain aresusceptible to inhibition by the subject compounds.

In some embodiments, the subject compounds inhibit a PI4-kinase, asdetermined by an inhibition assay, e.g., by an assay that determines thelevel of activity of the enzyme either in a cell-free system or in acell after treatment with a subject compound, relative to a control, bymeasuring the IC₅₀ or EC₅₀ value, respectively. In certain embodiments,the subject compounds have an IC₅₀ value (or EC₅₀ value) of 10 μM orless, such as 3 μM or less, 1 μM or less, 500 nM or less, 300 nM orless, 200 nM or less, 100 nM or less, 50 nM or less, 30 nM or less, 10nM or less, 5 nM or less, 3 nM or less, 1 nM or less, or even lower.

In some embodiments, the subject compounds inhibit a PI4-kinase, asdetermined by a kinase activity assay, e.g., by an assay that determinesthe level of incorporation of radiolabeled phosphate from [γ-³²P]-ATPinto a substrate molecule after treatment with a subject compound,relative to a control, by measuring the beta-particle emission rateusing a scintillation counter or phosphorimaging. In certainembodiments, the subject compounds have an IC₅₀ value for PI4K-IIIβ ofless than about 1 μM, less than about 0.2 μM, less than about 0.1 μM,less than about 10 nM, less than about 1 nM, or even less, such asdescribed in Tables 2-3. In certain embodiments, the subject compoundshave an IC₅₀ value for PI4K-IIIα of less than about 50 μM, less thanabout 10 μM, less than about 1 μM, less than about 0.1 μM, less thanabout 10 nM, less than about 1 nM, or even less, such as described inTables 2-3. In certain further embodiments, the subject compounds havean IC50 value for PI4K-IIIβ of 50 μM or less, [etc., etc.], 10 nM orless, 6 nM or less, or even less, such as described in Tables 2-3. Incertain further embodiments, the subject compounds have an IC50 valuefor the PI3-kinase p110α-p85 complex of between about 8 and about 10 nM,between about 8 μM and about 10 μM, or even more, such as described inTables 2-3. In certain further embodiments, the subject compounds havean IC50 value for the PI3-kinase p110γ-p85 complex of from about 2 toabout 4 nM, of from about 4 μM to 5 μM, or even more, such as describedin Tables 2-3. In certain further embodiments, the subject compoundshave an IC50 value for the type II PI3-kinase beta of less than about 1μM, less than about 150 nM, less than about 30 nM, or even less, such asdescribed in Tables 2-3. In certain embodiments, the subject compoundshave an IC50 value for type II PI3-kinase alpha of less than 10 μM. Incertain further embodiments, more than one of the above criteria isindependently satisfied by a particular compound.

In some embodiments, the potency of the PI 4-kinase inhibiting compoundstrack with anti-infective (e.g., antiviral) activity. In some cases, theenzymatic and anti-infective activities of the subject compoundsdiverge. In some embodiments, the anti-infective activity of the subjectcompounds depends on a combination of inhibition of both PI4KIIIα andPI4KIIIβ, or a combination of inhibition of class III PI4-kinases andclass II PI3-kinases (especially class II PI3-kinase beta). The subjectcompound may have increased specificity for one isoform of thesePI-kinase family members.

In certain embodiments, the subject compounds have no significant effecton the viability of a mammalian cell, as determined by a cellcytotoxicity assay, e.g., as determined by administering a subjectcompound to a HeLa cell and determining the number of viable cellspresent. The subject compounds may exhibit a % cell viability, ascompared to a control (e.g., a DMSO control), of 15% or more, such as20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% ormore, 80% or more, 90% or more, 100% or more, 120% or more, or evenhigher. The subject compounds may exhibit a CC₅₀ value of 1 nM orhigher, such as 100 nM or higher, 300 nM or higher, 1 μM or higher, 3 μMor higher, 5 μM or higher, 10 μM or higher, 20 μM or higher, 30 μM orhigher, 50 μM or higher, or even higher.

In certain embodiments, the compounds have a therapeutic index (e.g.,the ratio of a compound's cytotoxicity (e.g., cell cytotoxicity, CC50)to bioactivity (e.g., antiviral activity, EC50)) that is 20 or more,such as 50 or more, 100 or more, 200 or more, 300 or more, 400 or more,500 or more, or even more.

As summarized above, aspects of the disclosure include methods ofinhibiting a PI-kinase (e.g., a PI3, a PI4-lIla, or a PI4-IIIβ kinase).A subject compound may inhibit at least one activity of the PI-kinase inthe range of 10% to 100%, e.g., by 10% or more, 20% or more, 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,or 90% or more. In certain assays, a subject compound may inhibit itstarget with an 10₅₀ of 1×10⁻⁶ M or less (e.g., 1×10⁻⁶ M or less, 1×10⁻⁷M or less, 1×10⁻⁸ M or less, 1×10⁻⁹ M or less, 1×10⁻¹⁹ M or less, or1×10⁻¹¹ M or less).

The protocols that may be employed in determining PI-kinase activity arenumerous, and include but are not limited to cell-free assays, e.g.,binding assays; assays using purified enzymes, cellular assays in whicha cellular phenotype is measured, e.g., gene expression assays; and invivo assays that involve a particular animal (which, in certainembodiments may be an animal model for a condition related to the targetpathogen).

In some embodiments, the subject method is an in vitro method thatincludes contacting a sample with a subject compound that specificallyinhibits a target PI-kinase. In certain embodiments, the sample issuspected of containing the PI-kinase and the subject method furthercomprises evaluating whether the compound inhibits the PI-kinase. Incertain embodiments, the PI-kinase is a PI4-kinase or a PI-3 kinase.

In certain embodiments, the subject compound is a modified compound thatincludes a label, e.g., a fluorescent label, and the subject methodfurther includes detecting the label, if present, in the sample, e.g.,using optical detection.

In certain embodiments, the compound is modified with a support or withaffinity groups that bind to a support (e.g. biotin), such that anysample that does not bind to the compound may be removed (e.g., bywashing). The specifically bound target PI-kinase, if present, may thenbe detected using any convenient means, such as, using the binding of alabeled target specific probe, or using a fluorescent protein reactivereagent.

In another embodiment of the subject method, the sample is known tocontain the target PI-kinase.

Methods

Contrary to the classic paradigm of anti-infective therapy, the presentdisclosure provides methods of treating pathogen infection by targetinga host function and/or molecule upon which the pathogen is dependent,thereby decreasing the ability of the pathogen to avoid the therapeuticagent by mutation. In addition, by utilizing such a target, the methodsof the disclosure allow combination therapies in which multiple targetsare addressed, thereby increasing the ability to eliminate theinfectious agent. The methods also provide a broad platform foranti-infective therapies by targeting a host function. In addition, incases where the pathogen encodes its own PI-kinase(s), the presentdisclosure provides methods of treating pathogen infection by targetingthe pathogen PI-kinase.

Pathogens of interest include those described in Glenn et al., “PIP-2Inhibition-Based Antiviral and Anti-Hyperlipidemic Therapies”WO2009/148541, the disclosure of which is herein incorporated byreference in its entirety. In some embodiments, the pathogen is selectedfrom HCV, rhinovirus (e.g., B or C), P. falciparum, Ebola virus,Francisella tularensis, hantavirus, vaccinia, smallpox, Japaneseencephalitis virus, hepatitis A virus, and influenza virus, PolioVirus,Enterovirus (e.g., A-D), West Nile Virus, and Dengue Virus (e.g., 1-4).

In some embodiments, where the pathogen is HCV, useful compounds includethose having a high first-pass effect and consequent low systemicbioavailability, which are targeted to the liver, and which aretypically discarded in early drug development. In other embodiments forthe treatment of HCV, the compound, or formulation, is modified forliver-specific targeting.

In some cases, the method is a method of inhibiting a PI4-kinase in asample.

As such, aspects of the method include contacting a sample with asubject compound (e.g., as described above) under conditions by whichthe compound inhibits the PI4-kinase. Any convenient protocol forcontacting the compound with the sample may be employed. The particularprotocol that is employed may vary, e.g., depending on whether thesample is in vitro or in vivo. For in vitro protocols, contact of thesample with the compound may be achieved using any convenient protocol.In some instances, the sample includes cells that are maintained in asuitable culture medium, and the complex is introduced into the culturemedium. For in vivo protocols, any convenient administration protocolmay be employed. Depending upon the potency of the compound, the cellsof interest, the manner of administration, the number of cells present,various protocols may be employed.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form,containing one or more components of interest.

In some embodiments, the subject method is a method of treating asubject for an infective disease. In some embodiments, the subjectmethod includes administering to the subject an effective amount of a2-aminophenylthiazole compound (e.g., as described above). In someembodiments, the infective disease condition results from infection witha positive-stranded RNA virus, negative stranded RNA virus, or a DNAvirus. In some embodiments, the infective disease condition results frominfection with a pathogen selected from the group of viral familiesconsisting of Picornaviridae, Flaviviridae, Filoviridae, Bunyaviridae,Poxyiridae, and Orthomyxoviridae. In some embodiments, the infectivedisease condition results from infection with a pathogen selected fromthe phylum Apicomplexa or from the order Kinetoplastida. In someembodiments, the infective disease condition results from infection witha bacterium. In some embodiments, the infective disease conditionresults from infection with a pathogen selected from the groupconsisting of HCV, rhinovirus (e.g., B or C), P. falciparum, Ebolavirus, Francisella tularensis, hantavirus, vaccinia, smallpox, Japaneseencephalitis virus, hepatitis A virus, and influenza virus, PolioVirus,Enterovirus (e.g., A-D), West Nile Virus, and Dengue Virus (e.g., 1-4).In some embodiments, the pathogen is HCV. In some embodiments, thepathogen is rhinovirus or P. falciparum. In some embodiments, thepathogen is hepatitis A virus.

In some embodiments, the pathogen is characterized by having a BAAPPdomain that interacts with PIP-2, or a protein that binds PI(4,5)P₂ orPI(4)P. In some embodiments, the pathogen is characterized by having aprotein that interacts with one or more PI-4 kinases or PI phosphatases.In some embodiments, the BAAPP domain is derived from NS5A or NS4Bprotein. In some embodiments, the infective disease condition is causedby infection of a pathogen susceptible to PI4-kinase inhibition. In someembodiments, the compound specifically inhibits the PI4-kinase. In someembodiments, the compound has broad spectrum activity against two ormore pathogens. In some embodiments, the compound modulates the activityof PIP-2. In some embodiments, the compound interferes with theinteraction of a BAAPP domain and PIP-2 of the pathogen. In someembodiments, the compound blocks pathogen replication.

In some embodiments, the subject method is a method of treating asubject for an elevated level of VLDL or LDL cholesterol. In someembodiments, the subject method includes administering to the subject aneffective amount of a 2-aminophenylthiazole compound (e.g., as describedabove), alone or in combination with other drugs known to affect LDL orVLDL levels (e.g., 3-hydroxy-3-methylglutaryl-coenzyme A reductaseinhibitors such as lovastatin, fluvastatin, atorvastatin, pravastatin,simvastatin, rosuvastatin, etc.; microsomal triglyceride transferprotein inhibitors such as lomitapide; inhibitors of intestinalcholesterol absorption such as ezetimibe; peroxisomeproliferator-activated receptor type alpha activators such asfenofibrate).

In some embodiments, the subject is human. In some embodiments, thecompound is administered as a pharmaceutical preparation.

In some embodiments, the subject method is a method of inhibiting viralinfection, the method including contacting virus-infected cells with aneffective dose of a 2-aminophenylthiazole compound (e.g., as describedabove) to inhibit viral replication. In some embodiments, the methodfurther includes contacting the cells with a second antiviral agent.

In some embodiments, the compound is formulated to be targeted to theliver.

In certain embodiments, the compound is a modified compound thatincludes a label, and the method further includes detecting the label inthe subject. The selection of the label depends on the means ofdetection. Any convenient labeling and detection systems may be used inthe subject methods, see e.g., Baker, “The whole picture,” Nature, 463,2010, p977-980. In certain embodiments, the compound includes afluorescent label suitable for optical detection. In certainembodiments, the compound includes a radiolabel for detection usingpositron emission tomography (PET) or single photon emission computedtomography (SPECT). In some cases, the compound includes a paramagneticlabel suitable for tomographic detection. The subject compound may belabeled, as described above, although in some methods, the compound isunlabelled and a secondary labeling agent is used for imaging.

Utility

The compounds and methods of the invention, e.g., as described herein,find use in a variety of applications. Applications of interest include,but are not limited to: research applications and therapeuticapplications. Methods of the invention find use in a variety ofdifferent applications including any convenient application whereinhibition of a PI4-kinase is desired.

The subject compounds and methods find use in a variety of researchapplications. The subject compounds and methods may be used in theoptimization of the bioavailability and metabolic stability ofcompounds.

The subject MCIPs and methods find use in a variety of therapeuticapplications. Therapeutic applications of interest include thoseapplications in which pathogen infection is the cause or a compoundingfactor in disease progression. As such, the subject compounds find usein the treatment of a variety of different conditions in which theinhibition and/or treatment of viral infection in the host is desired.For example, the subject MCIPs and methods may find use in treating apathogen caused infective disease such as HCV.

Pharmaceutical Compositions

The above-discussed compounds can be formulated using any convenientexcipients, reagents and methods. Compositions are provided informulation with a pharmaceutically acceptable excipient(s). A widevariety of pharmaceutically acceptable excipients are known in the artand need not be discussed in detail herein. Pharmaceutically acceptableexcipients have been amply described in a variety of publications,including, for example, A. Gennaro (2000) “Remington: The Science andPractice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins;Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Anselet al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In some embodiments, the subject compound is formulated in an aqueousbuffer. Suitable aqueous buffers include, but are not limited to,acetate, succinate, citrate, and phosphate buffers varying in strengthsfrom 5 mM to 100 mM. In some embodiments, the aqueous buffer includesreagents that provide for an isotonic solution. Such reagents include,but are not limited to, sodium chloride; and sugars e.g., mannitol,dextrose, sucrose, and the like. In some embodiments, the aqueous bufferfurther includes a non-ionic surfactant such as polysorbate 20 or 80.Optionally the formulations may further include a preservative. Suitablepreservatives include, but are not limited to, a benzyl alcohol, phenol,chlorobutanol, benzalkonium chloride, and the like. In many cases, theformulation is stored at about 4° C. Formulations may also belyophilized, in which case they generally include cryoprotectants suchas sucrose, trehalose, lactose, maltose, mannitol, and the like.Lyophilized formulations can be stored over extended periods of time,even at ambient temperatures. In some embodiments, the subject compoundis formulated for sustained release.

In some embodiments, the subject compound and an antiviral agent, e.g.interferon, ribavirin, Enfuvirtide; RFI-641(4,4″-bis-{4,6-bis-[3-(bis-carbamoylmethyl-sulfamoyl)-phenylamino]-(1,3,5)triazin-2-ylamino}-biphenyl-2,2″-disulfonic acid); BMS-433771(2H-Imidazo[4,5-c]pyridin-2-one,1-cyclopropyl-1,3-dihydro-3-((1-(3-hydroxypropyl)-1H-benzimidazol-2-yl)methyl));arildone; Pleconaril(3-(3,5-Dimethyl-4-(3-(3-methyl-5-isoxazolyl)propoxy)phenyl)-5-(trifluoromethyl)-1,2,4-oxadiazole);Amantadine (tricyclo[3.3.1.1.3,7]decane-1-amine hydrochloride);Rimantadine (alpha-methyltricyclo[3.3.1.1.3,7]decane-1-methanaminehydrochloride); Acyclovir (acycloguanosine); Valaciclovir; Penciclovir(9-(4-hydroxy-3-hydroxymethyl-but-1-yl)guanine); Famciclovir (diacetylester of 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)-6-deoxyguanine);Gancyclovir (9-(1,3-dihydroxy-2-propoxymethyl)guanine); Ara-A (adenosinearabinoside); Zidovudine (3′-azido-2′,3′-dideoxythymidine); Cidofovir(1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine dihydrate);Dideoxyinosine (2′,3′-dideoxyinosine); Zalcitabine(2′,3′-dideoxycytidine); Stavudine(2′,3′-didehydro-2′,3′-dideoxythymidine); Lamivudine((−)-β-L-3′-thia-2′,3′-dideoxycytidine); Abacavir(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsuccinate); Emtricitabine(−)-8-L-3′-thia-2′,3′-dideoxy-5-fluorocytidine); Tenofovir disoproxil(Fumarate salt of bis(isopropoxycarbonyloxymethyl) ester of(R)-9-(2-phosphonylmethoxypropyl)adenine); Bromovinyl deoxyuridine(Brivudin); Iodo-deoxyuridine (Idoxuridine); Trifluorothymidine(Trifluridine); Nevirapine(1′-cyclopropyl-5,1′-dihydro-4-methyl-6H-dipyrido[3,2-b:2′,3′-f][1,4]diazepin-6-one);Delavirdine(1-(5-methanesulfonamido-1H-indol-2-yl-carbonyl)-4-[3-(1-methylethyl-amino)pyridinyl)piperazine monomethane sulfonated); Efavirenz((−)6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one);Foscarnet (trisodium phosphonoformate); Ribavirin(1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide); Raltegravir(N-[(4-Fluorophenyl)methyl]-1,6-dihydro-5-hydroxy-1-methyl-2-[1-methyl-1-[[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino]ethyl]-6-oxo-4-pyrimidinecarboxamidemonopotassium salt); Neplanocin A; Fomivirsen; Saquinavir (SQ);Ritonavir([5S-(5R,8R,10R,11R)]-10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazamidecan-13-oicacid 5-th iazolylmethyl ester); Indinavir([(1S,2R,5(S)-2,3,5-trideoxy-N-(2,3-dihydro-2-hydroxy-1H-inden-1-yl)-5-[2-[[(1,1-dimethylethyl)amino]carbonyl]-4-pyridinylmethyl)-1-piperazinyl]-2-(phenylmethyl-erythro)pentonamide);Amprenavir; Nelfinavir; Lopinavir; Atazanavir; Bevirimat; Indinavir;Relenza; Zanamivir; Oseltamivir; Tarvacin; etc. are administered toindividuals in a formulation (e.g., in the same or in separateformulations) with a pharmaceutically acceptable excipient(s).

In another aspect of the present invention, a pharmaceutical compositionis provided, comprising, or consisting essentially of, a compound of thepresent invention, or a pharmaceutically acceptable salt, isomer,tautomer or prodrug thereof, and further comprising one or moreadditional anti-HCV therapeutic agents selected from the groupconsisting of: an HCV NS3 protease inhibitor, an HCV NS5B RNA-dependentRNA polymerase inhibitor, a thiazolide, a sustained release thiazolide,a nucleoside analog, an interferon-alpha or lambda, a pegylatedinterferon, ribavirin, levovirin, viramidine, a TLR7 agonist, a TLR9agonist, a cyclophilin inhibitor, an alpha-glucosidase inhibitor, anNS5A inhibitor, an NS3 helicase inhibitor, clemizole or clemizole analog(such as the benzimidizole and indazole analogs described in U.S. patentapplication Ser. Nos. 12/383,071 and 12/383,030), or other NS4Binhibitor including an NS4B amphipathic helix inhibitor. The subjectcompound and second antiviral agent, as well as additional therapeuticagents as described herein for combination therapies, can beadministered orally, subcutaneously, intramuscularly, parenterally, orother route. The subject compound and second antiviral agent may beadministered by the same route of administration or by different routesof administration. The therapeutic agents can be administered by anysuitable means including, but not limited to, for example, oral, rectal,nasal, topical (including transdermal, aerosol, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenousand intradermal), intravesical or injection into an affected organ.

In some embodiments, the subject compound and an antimalarial agent,e.g., chloroquine, primaquine, mefloquine, doxycycline,atovaquone-proguanil, quinine, quinidine, artesunate, artemether,lumefantrine; etc. are administered to individuals in a formulation(e.g., in the same or in separate formulations) with a pharmaceuticallyacceptable excipient(s). The subject compound and second antimalarialagent, as well as additional therapeutic agents as described herein forcombination therapies, can be administered orally, subcutaneously,intramuscularly, parenterally, or other route. The subject compound andsecond antimalarial agent may be administered by the same route ofadministration or by different routes of administration. The therapeuticagents can be administered by any suitable means including, but notlimited to, for example, oral, rectal, nasal, topical (includingtransdermal, aerosol, buccal and sublingual), vaginal, parenteral(including subcutaneous, intramuscular, intravenous and intradermal),intravesical or injection into an affected organ.

The subject compounds may be administered in a unit dosage form and maybe prepared by any methods well known in the art. Such methods includecombining the subject compound with a pharmaceutically acceptablecarrier or diluent which constitutes one or more accessory ingredients.A pharmaceutically acceptable carrier is selected on the basis of thechosen route of administration and standard pharmaceutical practice.Each carrier must be “pharmaceutically acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. This carrier can be a solid or liquid and thetype is generally chosen based on the type of administration being used.

Examples of suitable solid carriers include lactose, sucrose, gelatin,agar and bulk powders. Examples of suitable liquid carriers includewater, pharmaceutically acceptable fats and oils, alcohols or otherorganic solvents, including esters, emulsions, syrups or elixirs,suspensions, solutions and/or suspensions, and solution and orsuspensions reconstituted from non-effervescent granules andeffervescent preparations reconstituted from effervescent granules. Suchliquid carriers may contain, for example, suitable solvents,preservatives, emulsifying agents, suspending agents, diluents,sweeteners, thickeners, and melting agents. Preferred carriers areedible oils, for example, corn or canola oils. Polyethylene glycols,e.g. PEG, are also good carriers.

Any drug delivery device or system that provides for the dosing regimenof the instant disclosure can be used. A wide variety of deliverydevices and systems are known to those skilled in the art.

Although such may not be necessary, compounds and agents describedherein can optionally be targeted to the liver, using any knowntargeting means. The compounds of the disclosure may be formulated witha wide variety of compounds that have been demonstrated to targetcompounds to hepatocytes. Such liver targeting compounds include, butare not limited to, asialoglycopeptides; basic polyamino acidsconjugated with galactose or lactose residues; galactosylated albumin;asialoglycoprotein-poly-L-lysine) conjugates; lactosaminated albumin;lactosylated albumin-poly-L-lysine conjugates; galactosylatedpoly-L-lysine; galactose-PEG-poly-L-lysine conjugates;lactose-PEG-poly-L-lysine conjugates; asialofetuin; and lactosylatedalbumin.

The terms “targeting to the liver” and “hepatocyte targeted” refer totargeting of a compound to a hepatocyte, particularly a virally infectedhepatocyte, such that at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,or at least about 90%, or more, of the compound administered to thesubject enters the liver via the hepatic portal and becomes associatedwith (e.g., is taken up by) a hepatocyte.

HCV infection is associated with liver fibrosis and in certainembodiments the inhibitors may be useful in treating liver fibrosis(particularly preventing, slowing of progression, etc.). The methodsinvolve administering an compound of the disclosure as described above,in an amount effective to reduce viral load, thereby treating liverfibrosis in the subject. Treating liver fibrosis includes reducing therisk that liver fibrosis will occur; reducing a symptom associated withliver fibrosis; and increasing liver function.

Whether treatment with a compound as described herein is effective inreducing liver fibrosis is determined by any of a number ofwell-established techniques for measuring liver fibrosis and liverfunction. The benefit of anti-fibrotic therapy can be measured andassessed by using the Child-Pugh scoring system which comprises amulti-component point system based upon abnormalities in serum bilirubinlevel, serum albumin level, prothrombin time, the presence and severityof ascites, and the presence and severity of encephalopathy. Based uponthe presence and severity of abnormality of these parameters, patientsmay be placed in one of three categories of increasing severity ofclinical disease: A, B, or C.

Treatment of liver fibrosis (e.g., reduction of liver fibrosis) can alsobe determined by analyzing a liver biopsy sample. An analysis of a liverbiopsy comprises assessments of two major components: necroinflammationassessed by “grade” as a measure of the severity and ongoing diseaseactivity, and the lesions of fibrosis and parenchymal or vascularremodeling as assessed by “stage” as being reflective of long-termdisease progression. See, e.g., Brunt (2000) Hepatol. 31:241-246; andMETAVIR (1994) Hepatology 20:15-20. Based on analysis of the liverbiopsy, a score is assigned. A number of standardized scoring systemsexist which provide a quantitative assessment of the degree and severityof fibrosis. These include the METAVIR, Knodell, Scheuer, Ludwig, andIshak scoring systems.

The METAVIR scoring system is based on an analysis of various featuresof a liver biopsy, including fibrosis (portal fibrosis, centrilobularfibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis,acidophilic retraction, and ballooning degeneration); inflammation(portal tract inflammation, portal lymphoid aggregates, and distributionof portal inflammation); bile duct changes; and the Knodell index(scores of periportal necrosis, lobular necrosis, portal inflammation,fibrosis, and overall disease activity). The definitions of each stagein the METAVIR system are as follows: score: 0, no fibrosis; score: 1,stellate enlargement of portal tract but without septa formation; score:2, enlargement of portal tract with rare septa formation; score: 3,numerous septa without cirrhosis; and score: 4, cirrhosis.

Knodell's scoring system, also called the Hepatitis Activity Index,classifies specimens based on scores in four categories of histologicfeatures: I. Periportal and/or bridging necrosis; II. Intralobulardegeneration and focal necrosis; III. Portal inflammation; and IV.Fibrosis. In the Knodell staging system, scores are as follows: score:0, no fibrosis; score: 1, mild fibrosis (fibrous portal expansion);score: 2, moderate fibrosis; score: 3, severe fibrosis (bridgingfibrosis); and score: 4, cirrhosis. The higher the score, the moresevere the liver tissue damage. Knodell (1981) Hepatol. 1:431.

In the Scheuer scoring system scores are as follows: score: 0, nofibrosis; score: 1, enlarged, fibrotic portal tracts; score: 2,periportal or portal-portal septa, but intact architecture; score: 3,fibrosis with architectural distortion, but no obvious cirrhosis; score:4, probable or definite cirrhosis. Scheuer (1991) J. Hepatol. 13:372.

The Ishak scoring system is described in Ishak (1995) J. Hepatol.22:696-699. Stage 0, No fibrosis; Stage 1, Fibrous expansion of someportal areas, with or without short fibrous septa; stage 2, Fibrousexpansion of most portal areas, with or without short fibrous septa;stage 3, Fibrous expansion of most portal areas with occasional portalto portal (P-P) bridging; stage 4, Fibrous expansion of portal areaswith marked bridging (P-P) as well as portal-central (P-C); stage 5,Marked bridging (P-P and/or P-C) with occasional nodules (incompletecirrhosis); stage 6, Cirrhosis, probable or definite.

In some embodiments, a therapeutically effective amount of a compound ofthe disclosure is an amount of compound that effects a change of oneunit or more in the fibrosis stage based on pre- and post-therapymeasures of liver function (e.g, as determined by biopsies). Inparticular embodiments, a therapeutically effective amount of thesubject compound reduces liver fibrosis by at least one unit in theChild-Pugh, METAVIR, the Knodell, the Scheuer, the Ludwig, or the Ishakscoring system.

Secondary, or indirect, indices of liver function can also be used toevaluate the efficacy of treatment. Morphometric computerizedsemi-automated assessment of the quantitative degree of liver fibrosisbased upon specific staining of collagen and/or serum markers of liverfibrosis can also be measured as an indication of the efficacy of asubject treatment method. Secondary indices of liver function include,but are not limited to, serum transaminase levels, prothrombin time,bilirubin, platelet count, portal pressure, albumin level, andassessment of the Child-Pugh score. An effective amount of the subjectcompound is an amount that is effective to increase an index of liverfunction by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, or at leastabout 80%, or more, compared to the index of liver function in anuntreated individual, or to a placebo-treated individual. Those skilledin the art can readily measure such indices of liver function, usingstandard assay methods, many of which are commercially available, andare used routinely in clinical settings.

Serum markers of liver fibrosis can also be measured as an indication ofthe efficacy of a subject treatment method. Serum markers of liverfibrosis include, but are not limited to, hyaluronate, N-terminalprocollagen III peptide, 7S domain of type IV collagen, C-terminalprocollagen I peptide, and laminin. Additional biochemical markers ofliver fibrosis include α-2-macroglobulin, haptoglobin, gamma globulin,apolipoprotein A, and gamma glutamyl transpeptidase.

A therapeutically effective amount of the subject compound is an amountthat is effective to reduce a serum level of a marker of liver fibrosisby at least about 10%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, or at least about80%, or more, compared to the level of the marker in an untreatedindividual, or to a placebo-treated individual. Those skilled in the artcan readily measure such serum markers of liver fibrosis, using standardassay methods, many of which are commercially available, and are usedroutinely in clinical settings. Methods of measuring serum markersinclude immunological-based methods, e.g., enzyme-linked immunosorbentassays (ELISA), radioimmunoassays, and the like, using antibody specificfor a given serum marker.

Qualitative or quantitative tests of functional liver reserve can alsobe used to assess the efficacy of treatment with an agent. Theseinclude: indocyanine green clearance (ICG), galactose eliminationcapacity (GEC), aminopyrine breath test (ABT), antipyrine clearance,monoethylglycine-xylidide (MEG-X) clearance, and caffeine clearance.

As used herein, a “complication associated with cirrhosis of the liver”refers to a disorder that is a sequellae of decompensated liver disease,i.e., or occurs subsequently to and as a result of development of liverfibrosis, and includes, but it not limited to, development of ascites,variceal bleeding, portal hypertension, jaundice, progressive liverinsufficiency, encephalopathy, hepatocellular carcinoma, liver failurerequiring liver transplantation, and liver-related mortality.

A therapeutically effective amount of a compound in this context can beregarded as an amount that is effective in reducing the incidence (e.g.,the likelihood that an individual will develop) of a disorder associatedwith cirrhosis of the liver by at least about 10%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, or at least about 80%, or more, compared to an untreatedindividual, or to a placebo-treated individual.

Whether treatment with the subject compound is effective in reducing theincidence of a disorder associated with cirrhosis of the liver canreadily be determined by those skilled in the art.

Reduction in HCV viral load, as well as reduction in liver fibrosis, canbe associated with an increase in liver function. Thus, the disclosureprovides methods for increasing liver function, generally involvingadministering a therapeutically effective amount of a compound of thedisclosure. Liver functions include, but are not limited to, synthesisof proteins such as serum proteins (e.g., albumin, clotting factors,alkaline phosphatase, aminotransferases (e.g., alanine transaminase,aspartate transaminase), 5′-nucleosidase, γ-glutaminyltranspeptidase,etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesisof bile acids; a liver metabolic function, including, but not limitedto, carbohydrate metabolism, amino acid and ammonia metabolism, hormonemetabolism, and lipid metabolism; detoxification of exogenous drugs; ahemodynamic function, including splanchnic and portal hemodynamics; andthe like.

Whether a liver function is increased is readily ascertainable by thoseskilled in the art, using well-established tests of liver function.Thus, synthesis of markers of liver function such as albumin, alkalinephosphatase, alanine transaminase, aspartate transaminase, bilirubin,and the like, can be assessed by measuring the level of these markers inthe serum, using standard immunological and enzymatic assays. Splanchniccirculation and portal hemodynamics can be measured by portal wedgepressure and/or resistance using standard methods. Metabolic functionscan be measured by measuring the level of ammonia in the serum.

Whether serum proteins normally secreted by the liver are in the normalrange can be determined by measuring the levels of such proteins, usingstandard immunological and enzymatic assays. Those skilled in the artknow the normal ranges for such serum proteins. The following arenon-limiting examples. The normal range of alanine transaminase is fromabout 7 to about 56 units per liter of serum. The normal range ofaspartate transaminase is from about 5 to about 40 units per liter ofserum. Bilirubin is measured using standard assays. Normal bilirubinlevels are usually less than about 1.2 mg/dL. Serum albumin levels aremeasured using standard assays. Normal levels of serum albumin are inthe range of from about 35 to about 55 g/L. Prolongation of prothrombintime is measured using standard assays. Normal prothrombin time is lessthan about 4 seconds longer than control.

A therapeutically effective amount of a compound in this context is onethat is effective to increase liver function by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, ormore. For example, a therapeutically effective amount of a compound isan amount effective to reduce an elevated level of a serum marker ofliver function by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or more, or to reduce the level ofthe serum marker of liver function to within a normal range. Atherapeutically effective amount of a compound is also an amounteffective to increase a reduced level of a serum marker of liverfunction by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more, or to increase the level of theserum marker of liver function to within a normal range.

HCV infection is associated with hepatic cancer and in certainembodiments the present disclosure provides compositions and methods ofreducing the risk that an individual will develop hepatic cancer. Themethods involve administering the subject compound, as described above,wherein viral load is reduced in the individual, and wherein the riskthat the individual will develop hepatic cancer is reduced. An effectiveamount of a compound is one that reduces the risk of hepatic cancer byat least about 10%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, or more. Whether the risk of hepaticcancer is reduced can be determined in, e.g., study groups, whereindividuals treated according to the subject methods have reducedincidence of hepatic cancer.

Subjects Amenable to Treatment Using the Compounds of the Disclosure

Individuals who have been clinically diagnosed as infected with apathogen of interest are suitable for treatment with the methods of thepresent disclosure. In particular embodiments of interest, individualsof interest for treatment according to the disclosure have detectablepathogen titer indicating active replication, for example an HCV titerof at least about 10⁴, at least about 10⁵, at least about 5×10⁵, or atleast about 10⁶, or greater than 2 million genome copies of HCV permilliliter of serum. Similar methods may be used to determine whethersubjects infected with another pathogen are suitable for treatment usingthe subject methods.

The effectiveness of the anti-infective treatment may be determinedusing any convenient method. For example, whether a subject method iseffective in treating a virus infection can be determined by measuringviral load, or by measuring a parameter associated with infection.

Viral load can be measured by measuring the titer or level of virus inserum. These methods include, but are not limited to, a quantitativepolymerase chain reaction (PCR) and a branched DNA (bDNA) test. Manysuch assays are available commercially, including a quantitative reversetranscription PCR (RT-PCR) (Amplicor HCV Monitor™, Roche MolecularSystems, New Jersey); and a branched DNA (deoxyribonucleic acid) signalamplification assay (Quantiplex™ HCV RNA Assay (bDNA), Chiron Corp.,Emeryville, Calif.). See, e.g., Gretch et al. (1995) Ann. Intern. Med.123:321-329.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use embodiments of the present disclosure, and are not intendedto limit the scope of what the inventors regard as their invention norare they intended to represent that the experiments below are all or theonly experiments performed. Efforts have been made to ensure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the claims appended hereto.

Example 1 Synthesis and Assays

In the assays described here, p110α-p85 complex and p110γ were acquiredfrom Millipore. Assays were performed with La-phosphatidylinositol(Avanti) as described in Knight et al. Nat. Protoc. 2007; 2(10):2459-66.Inhibitor series were prepared 10% DMSO as 5× stocks for the assay.Assays of HsVps34 were performed as described in Knight et al, exceptthat the final assay buffer composition used was changed to 20 mM HEPES7.5, 100 mM NaCl, 3 mM MgCl, 1 mg/mL PI and 44 nM hvps34 was used in theassay. For inhibitors with an apparent IC50 less than or equal to 22 nM,values were reassayed using 4.4 nM hvps34 with 3 mM MnCl2. COS-7 cellswere cultured in 10 cm dishes and transfected at 70% confluence with 10μg of plasmid DNA (HA-tagged bovine PI4KIIIβ, HA-tagged human PI4KIIIα)using Lipofectamine 2000 and 5 ml Opti-MEM following the manufacturer'sinstructions. After 5 hours the transfection medium was replaced with 10ml complete DMEM. 36 hrs post transfection, cells were washed once with5 ml PBS (pH 7.4) and lysed in 1 ml of lysis buffer (1) on ice. Lysateswere collected by scraping and after 15 min they were centrifuged at13,000 rpm for 10 min. To the lysates was added 200 μl of protein GSepharose 4 fast flow beads that were prewashed with PBS and lysisbuffer and 2 μg of anti-HA antibody. The tubes were then incubatedovernight at 4 degrees C. in a tube rotator. The Sepharose beads in thelysate were washed twice with 150 mM NaCl in RIPA buffer, twice withRIPA buffer and once with kinase buffer (50 mM Tris/HCl, pH 7.5, 20 mMMgCl2, 1 mM EGTA, 1 mM Ptdlns, 0.4% Triton X-100, 0.5 mg/mL BSA) andfinally the beads were resuspended in 200 μL kinase buffer. Kinasereactions were run in a mixture of 45 μL of PI buffer (1 mM PI in kinasebuffer), 10 μL of immunoprecipitated beads, 2 μL of inhibitors(dissolved and diluted in DMSO) or DMSO and 5 μL of [γ-³²P]-ATP (1 mMand 2 μCi/tube). The immunoprecipitates in PI buffer were pre-incubatedwith the drugs for 20 min prior the initiation of kinase reaction byadding ATP and the reactions were carried out for 30 min in 15 mlpolypropolyne tubes. Reactions were terminated by addition of 3 ml ofCHCl₃:CH₃OH:HCl (200:100:0.75) followed by 0.6 ml of 0.6N HCl to inducephase separation. The mixtures were vortexed, centrifuged at 2000 rpmfor 2 min and the upper phase was discarded. To the lower phase wasadded 1.5 ml of CHCl₃:CH₃OH:0.6N HCl (3:48:47) and the mixture vortexedand centrifuged at 2000 rpm for 2 min. The lower phase was thentransferred to counting vials and evaporated. Samples were counted in ascintillation beta counter after adding 5 ml of Instafluor(Perkin-Elmer).

Synthesis:

Compounds may be synthesized using any convenient method. For example,by similar methods to those described by Shokat et al. “Apharmacological map of the PI3-K family defines a role for p110alpha ininsulin signaling.” Cell. 2006; 125(4):733-47. Starting materials areobtained from Aldrich or Alfa Aesar. Reactions are monitored by LC/MSand reaction products characterized by LC/MS and ¹H NMR. Intermediatesand final products are purified by silica gel chromatography or byreverse phase HPLC.

PI-Kinase Assay:

Compounds are tested in C.1.1. PI kinase assays as described by Shokatet al., “A membrane capture assay for lipid kinase activity.” Nat.Protoc. 2007; 2(10):2459-66.

Anti-HCV Assay:

Anti-HCV assays are performed as described by Cho et al. “Identificationof a class of HCV inhibitors directed against the nonstructural proteinNS4B.” Sci. Transl. Med. 2011; 2(15):15ra6.

Broad-Spectrum Anti-Infective Assays:

Compounds are tested for activity against selected agents harboringproteins with BAAPP domains, or other PI-4 or PIP2 binding motifs, (i.e.Vaccinia virus, Japanese encephalitis virus, hepatitis A virus, andinfluenza virus) in clinical studies. Activity against multiple NIAIDCategory A, B, and C pathogens is assayed.

Vaccinia Virus Assay:

Standard plaque assays are performed on CV-1 cells, using methodsdescribed by Glenn et al., “Amphipathic helix-dependent localization ofNS5A mediates hepatitis C virus RNA replication.” J. Virol. 2003;77(10):6055-61, in the presence of vehicle or vehicle plus variousconcentrations of compound.

HAV Assay:

Huh7 cells harboring HAV replicons encoding a blasticidin resistancegene (Yang et al., “Disruption of innate immunity due to mitochondrialtargeting of a picornaviral protease precursor.” Proc Natl Acad Sci USA2007; 104(17):7253-8) is grown in media containing blasticidin, with orwithout various concentrations of compound. Anti-HAV activity isassessed by both cell plating efficiency and HAV RNA levels usingquantitative RT-PCR assays. A luciferase-linked HAV replicon fortranisient replication assays is used to evaluate the effects of HAVBAAPP domain mutants.

JEV Assay:

JEV assays are performed using both infectious virus in cell culture, aswell as in an in vivo animal model, using similar methods to thosedescribed by Shah et al. “Molecular characterization of attenuatedJapanese encephalitis live vaccine strain ML-17.” Vaccine. 2006;24(4):402-11.

Influenza Virus Assay:

Influenza virus assays are performed using infectious virus in cellculture, using similar methods to those described by Hossain et al.“Establishment and characterization of a Madin-Darby canine kidneyreporter cell line for influenza A virus assays.” J. Clin. Microbiol.48(7):2515-23.

Plasmodium falciparum Assay:

Plasmodium falciparum assays are performed using an erythrocyte-fedculture of P. falciparum ring forms, using similar methods to thosedescribed by Deu et al. “Functional Studies of Plasmodium falciparumDipeptidyl Aminopeptidase I Using Small Molecule Inhibitors and ActiveSite Probes.” Chemistry & Biology 17, 808-819.

Rhinovirus Assay:

Rhinovirus assays are performed by determining to what extent thecompound protects HeLa S3 cells from the cytopathic effect of aninoculum of human rhinovirus 14, using similar methods to thosedescribed by Buckwold et al., “Synergistic In Vitro Interactions betweenAlpha Interferon and Ribavirin against Bovine Viral Diarrhea Virus andYellow Fever Virus as Surrogate Models of Hepatitis C VirusReplication,” Antimicrobial Agents and Chemotherapy 47(7), 2293-2298.

HAV Assay:

HAV assays are performed by co-culturing Huh7-derived cells harboringthe blasticidin-selectable HAV replicon (HAV-Bla, described by Yang etal, “Disruption of innate immunity due to mitochondrial targeting of apicornaviral protease precursor”, PNAS 104(17), 7253-7258) for over twoweeks in DMEM with 10% FBS, 1% Pen-Strep, 1% L-Glutamine, 1%nonessential amino acids, and 4 μg/mL blasticidin, with variousconcentrations of compound or vehicle control in 6-well plates at adensity of 1000 HAV-Bla cells per well and 1/72 confluent plate worth ofHuh7 feeder cells per well. At the end of this culture, large coloniesin each well are counted and an effective concentration at which platingefficiency is decreased by 50% (EC50) is calculated. For compound PT423described below, the EC50 is below 50 nM.

For all of the assays described above, EC50, EC90, and CC50 values aredetermined, and experiments are performed starting drug treatments atvarious times post initiation of infection to help localize the mostsensitive aspects of each pathogen's life cycle to PI 4-kinaseinhibition.

Resistance Assays:

The capacity for emergence of resistance and its nature is determinedusing any convenient methods, for example methods that involvesequencing of any resistant isolates that are able to be propagated.Co-treatments with other drugs are also performed. Experiments areconducted under BL2+ conditions where appropriate.

TABLE 2 IC₅₀ IC₅₀ Com- IC₅₀ PI3K IC₅₀ PI3K PI3K PI3K EC₅₀ EC₅₀ CC₅₀ CC₅₀pound 110a 110g C2a C2b IC₅₀ IIIa IC₅₀ IIIb EC₅₀ 2a HRV14 P. falcip.Huh7.5 HeLa PIK93  31 nM    5 nM  >10 μM 440 nM    2 μM    24 nM   1 μM   3 μM PT17  87 nM   12 nM 200 nM  <30 nM 6.8 μM 8.38 nM   12 μM  >5μM  PT18 210 nM   49 nM 800 nM  <30 nM   1 μM   13 μM PT19  >10 μM   1.7μM  >10 μM >10 μM   5.7 μM    25 nM 6.54 μM  >20 μM PT110    1 μM   560nM  >10 μM >10 μM   6.2 μM  21.1 nM 8.5 μM   13 μM PT111  4.26 μM  2.14μM    3 μM  <30 nM   2 μM    6 μM PT27 516 nM   349 nM    5 μM    37 nM 10 μM >20 μM PT28 1.342 μM 1.067 μM 300 nM   200 nM   3 μM    5 μM PT298.612 μM  >10 μM     1 μM   300 nM  11 μM >20 μM PT210  2.05 μM  4.43 μM  1.7 μM    29 nM  14 μM >20 μM PT211 7.077 μM    1 μM    50 nM   7 μM   5 μM PT44 184 nM   95 nM  >30 μM   134 nM  >5 μM  92.2 nM  >5 μM PT45 74 nM   11 nM  >30 μM   141 nM   3 μM   10 μM PT46 100 nM    7 nM  >30μM   130 nM   5 μM  >5 μM PT47 136 nM    8 nM  >30 μM   636 nM  >5 μM  >5 μM PT48  55 nM    8 nM  10.4 μM   107 nM   3 μM  >5 μM PT410  16 nM   6 nM  >30 μM    65 nM   5 μM  >5 μM PT411  41 nM   11 nM  >30 μM  451 nM  >5 μM   >5 μM PT412   18 nM   4.6 μM    50 nM   5 μM    5 μMPT414 209 nM   70 nM   2.7 μM  35.2 nM 1.8 mM 37.3 nM >20 μM PT416  41nM    3 nM  >10 μM  27 nM   7.1 μM  44.9 nM 1.8 mM >20 μM PT417 196 nM  40 nM 3.30 μM   20 μM PT418  63 nM    5 nM 749 nM    29 nM 2.17 μM 3.9 μM PT419  9 nM    3 nM 749 nM  12.5 nM 13.0 nM  9.2 μM PT420  50 nM   8 nM    9 μM    71 nM 2.52 μM   12 μM PT421  58 nM    5 nM    22 μM  132 nM 4.89 μM   20 μM PT422  >10 μM 645 nM  26.7 μM   235 nM 2.14μM >20 μM PT423  65 nM   64 nM  >10 μM  28 nM  1.75 μM    7 nM  365 nM320 pM 3.20 nM >20 μM 12.6 μM <50 nM PT424  >10 μM  95 nM   2.5 μM  36.8nM  776 nM 7.01 nM  156 nM >20 μM PT425  49 nM   78 nM   7.8 μM 132 nM511 nM    8 nM  906 nM  13.6 μM  PT426  3.36 μM  19.1 nM 2.10 μM  19.8μM  PT427  20 nM   10 nM  1.97 μM  20 nM 292 nM  6.08 nM  434 nM  7.1 μMPT428 2.99 μM   20 μM PT429  89 nM    6 nM  4.16 μM    26 nM 1.74 μM >20μM

Humanized Mouse Model:

The performance characteristics of the compounds are assessed by dosingthe compounds in a mouse model with a humanized liver to determine theirin vivo pharmacokinetic (PK) and pharmacodynamic properties. This modelconsists of immunodeficient NOG mice (NOD/shi SCID II2rg −/−) harboringa Herpes virus-derived thymidine kinase (TK) transgene under the controlof an albumin promoter (Hasegawa et al., “The reconstituted ‘humanizedliver’ in TK-NOG mice is mature and functional.” Biochem Biophys ResCommun. 2011; 405(3):405-10). A brief exposure to ganciclovir targetsdestruction of the endogenous mouse liver, which is followed by thetransplantation of human liver cells. High level engraftment of humanhepatocytes can be achieved and efficient HCV infection established. Aquantitative analysis of in vivo PK parameters and efficacy of thecompounds and metabolites in the plasma of the humanized mice isperformed.

PK and PD:

Cohorts of humanized TK-NOG mice (e.g. 5 mice per treatment group) aregavaged with one dose of compound. Doses are chosen so as to maintain aconcentration above the respective EC50s. Serial aliquots of plasma areobtained at baseline, 15 minutes, 30 minutes, 1 hr and 2 hr post dosing.Similarly treated groups of mice are sacrificed to analyze levels of thedrugs and key metabolites in the liver. Concentrations of compounds andtheir metabolites are measured. PK parameters, such as C_(max), T½, AUC,and oral clearance are determined. Based on these parameters, cohorts ofhumanized TK-NOG mice (5 mice per treatment group) infected with HCVinoculums consisting of the infectious 2a clone (25) or de-identifiedpatient-derived sera are gavaged (Glenn et al., “In vivo antiviralefficacy of prenylation inhibitors against hepatitis delta virus (HDV).”Journal of Clinical Investigation. 2003; 112(3):407-14) for multipledoses and serial serum aliquots are obtained and antiviral efficacydetermined by measuring HCV titers by quantitative real-time PCR.Individually-treated mice can also serve as their own control whereinthe HCV titers before, during, and after treatment can be used to assessantiviral efficacy wherein an antiviral effect in indicated by a drop intiter during the treatment phase compared to the pretreatment phase,with (in the case where the virus has not been completely eliminatedduring the treatment period) or without (in the case where the virus hasbeen completely eliminated during the treatment period) an increase intiter following cessation of treatment.

Assessment of Drug Resistance:

(In vitro) Huh7 cells harboring a bicistronic genotype 1b subgenomicreplicon, wherein the first cistron encodes theneomycinphosphotransferase gene (which confers resistance to G418) andthe second cistron encodes the HCV non-structural proteins required forRNA genome replication, are grown in media containing G418 plusincreasing concentrations of compounds to select for drug resistantcolonies. This, along with extraction of the replicons harbored in theresistant cells, sequencing to identify candidate resistance mutations,and cloning of these mutations back into a wild-type replicon to confirmthey are truly causative of the resistance, is performed usingconvenient methods. In vivo) Inoculums consist of the infectious 2aclone and de-identified patient-derived sera. Once establishment ofinfection has been confirmed, humanized mice are treated by oral gavagewith a resistance-promoting regimen of compounds involving progressivedose escalation from a low dose (0.1 mg/kg/day), with drug holidays.Serum samples for analysis are taken at time 0, and serially thereafteron a weekly basis. The focus is first on any samples that display arebound in titer of greater than 1 log after a previous nadir. StandardDNA sequencing of individual clones isolated from RT-PCR cloning isperformed. Ultradeep pyrosequencing is reserved to determine earliestevidence of any observed resistance. As a control, similar experimentswith an HCV NS3 protease inhibitor (e.g. Boceprevir) are perfomed.

Example 2 Activity of Compound PT423

PT423 exhibits activity against both PI4KIIIα (IC50 1.75 micromolar) andPI4KIIIβ (IC50 7 nanomolar) in in vitro enzyme assays.

To further confirm its mechanism of action the effect of PT423 on PI-4levels was determined in cells using immunofluorescence to monitor theintracellular localized pools of PI-4. The effect of PT423 onintracellular pools of PIP2 was tested, which were predicted to besimilarly sensitive to inhibition using PT423. Indeed, as shown in FIG.1, a dose-dependent decrease in PI-4 and PIP2 was observed upontreatment with PT423. As a control for non-specific effects on thecells, the latter were also co-stained with an antibody to ER-localizedcalnexin, which revealed that the effect on PI-4 and PIP2 occurred inthe absence of any detectable effects on the ER.

FIG. 1 illustrates that PT423 decreases PI(4)P (top panels) andPI(4,5)P₂ (bottom panels) in a dose-dependent manner. Left panels:uninfected Huh7.5 cells; right panels: HCV-infected Huh7.5 cells. Cellswere treated with the indicated concentrations of PT423 or vehiclecontrol, and analyzed by immunofluorescence with antibodies to PI(4)P orPI(4,5)P₂ (green) along with an antibody to calnexin (red) to controlfor ER morphology.

The concentrations at which significant effects on PI-4 and PIP2 wereobserved in the target cells parallel the observed EC50 of 365 nM(CC50>10 micromolar) for PT423 against HCV in standard replicationassays(Cho et al., “Identification of a class of HCV inhibitors directedagainst the nonstructural protein NS4B.” Sci. Transl. Med. 2011;2(15):15ra6). Consistent with its host PI 4-kinase target, resistancewas not able to be selected using the HCV replicon system.

Example 3 Microsome Metabolites Study of Compound PT423

To determine the major metabolites of the compound, the compound isincubated with microsomes and analyzed using methods similar to thosedescribed by Guo et al., “In silico pharmacogenetics of warfarinmetabolism.” Nat. Biotechnol. 2006; 24(5):531-6. The followingmetabolites M1-M3 were identified in human microsomes after treatmentwith compound PT423.

Analytical and MS/MS Fragmentation analysis showed that the moleculebehaves well in LC/MS. It gives a strong M+H. It is fairly well detectedby UV at 280 nm. Its MS/MS is quite simple with diagnostic peaks at m/z392.

Metabolites: M1 is due to monohydroxylation (ES1, MS2: selected ions m/z504.1203, 392.0736). The 0 is clearly on the cyclopentyl group. M2 isthe desacyl metabolite (ES1, MS2: selected ions m/z 392.0716, 219.0578,191.0619). M3 is the metabolite formed via N-dearylation (ES1, MS2:selected ions m/z 300.0468).

Example 4

Standard medicinal chemistry quantitative structure-activityrelationship (QSAR) approaches are used to explore analogs of PT423systematically. PT423 exhibits sub-micromolar potency and thus wouldlikely be active in vivo at concentrations achievable with ahalf-life >30 minutes in animal models. A complementary strategy forimproving its activity in vivo involves increasing its biochemicalpotency to reduce its minimum effective concentration.

The N-phenylsulfonamide and acylamide portions of PT423-like compoundsare identified as the critical features driving selectivity and potency.A Topliss-informed strategy is used to gradually increase stericmodification to determine the steric/electronic/hydrophobicconfiguration optimal in each position to improve potency against PI4-kinase in biochemical assays. Scheme shown below.

For the acylamine position (Scheme A), analysis is already informed byexisting SAR data showing that large aliphatic substituents arenecessary for selectivity versus protein kinases, and correspondinglythe scheme is determines whether hydrophobic (preferred cyclobutyl) orsteric/electronic (preferred cyclohexyl) considerations are moreimportant for driving potency with substitution at this position. Forthe N-phenylsulfonamide position, SAR data is less constrained (otherthan suggesting the utility of phenyl substituents), and correspondinglythe scheme (Scheme B) is a typical aromatic Topliss scheme directedtoward determining the optimal steric, electronic, and hydrophobicityconfiguration with the minimal number of modifications from the parentcompound, with a phenyl substituent in the sulfonamide position. Such ascheme will also determine whether the para-hydroxy of PT423 serves anyessential function. Modifications found by these schemes resulting inimproved potency at either position are combined to see if an additiveor synergistic increase in potency is achievable.

Increased potency against PI 4-kinase activity may track with increasedantiviral activity, as it has to date. Such tracking will provideevidence that the increased antiviral activity is due to ourhypothesized mechanism of action. At some point the enzymatic andantiviral activities may diverge. This could be an indication thatmaximal antiviral activity depends on some combination of inhibition ofboth PI4KIIIα and PI4KIIIβ that might be lost as our analogs acquireincreased specificity for one of these PI 4-kinase family members.Alternatively, this could suggest that a new antiviral activity hasbegun to be acquired, such as generating a derivative of our lead thatbegins to exhibit anti-NS5B polymerase activity by virtue of acquiringfeatures present in nucleoside analogs or non-nucleoside inhibitors ofNS5B.

Example 5

The compounds of Table 1 were prepared according to the methodsdescribed above. Table 3 shows the results of testing selected compoundsfor anti-viral (EC₅₀) activity in a HCV genotype 2a in Huh7.5 cells byluciferase reporter assay, for cell toxicity (CC₅₀) and metabolichalflife (t½), according to the methods described above.

TABLE 3 CC₅₀ HLM t_(1/2) Cmpd # 2a EC₅₀ (μM) (min) AB3663 +++ 27.5AB3664 ++ 58 28.8 AB3680 + 54 AB3737 + 50 46.5 AB3755 + 23.6 AB3756 + 44158 AB3826 +++ AB3832 + 46 11.1 AB3835 ++ 48 28.7 AB3837 ++ 22 AB3862 ++22 27.3 AB3864 ++ AB3867 ++ 41 33.1 AB3913 + 59 24.2 AB3946 ++ AB3948 ++AB3950 +++ 12 AB3957 ++ 9 AB3977 ++ AB3979 ++ AB4021 +++ 13 3.9 AB4022 +21 21.9 AB4024 ++ AB4025 ++ 13 AB4050 ++ AB4051 ++ AB4052 +++ AB4053 ++AB4054 ++ AB4098 ++ 17.9 AB4099 +++ 17.6 AB4100 +++ 16.6 17.7 AB4106 ++58.5 AB4107 ++ AB4108 ++ AB4109 ++ 20.7 AB4110 ++ AB4111 ++ AB4112 ++AB4113 ++ AB4114 ++ AB4115 ++ AB4116 ++ AB4139 ++ AB4140 ++ AB4141 ++AB4145 ++ AB4162 ++ AB4163 ++ AB4164 ++ AB4165 ++ AB4166 ++ AB4171 ++AB4173 + AB4174 + AB4178 + AB4179 + AB4185 + AB4225 ++ AB4226 ++AB4227 + AB4228 + AB4229 + AB4237 + AB4239 ++ AB4240 + AB4241 + AB4242++ AB4243 ++ +++: EC₅₀ < 0.2 μM ++: 0.2 μM < EC₅₀ < 1 uM +: EC₅₀ > 1 μM

1. A compound described by the structure:

wherein: Z¹ and W are each independently a covalent bond or a linkingfunctional group; R¹ is selected from an alkyl, an aryl, analkyl-heterocycle and a heterocycle; R² is selected from hydrogen, ahalogen, an alkyl and an alkoxy; R³ is selected from hydrogen and analkyl; R⁴ is selected from an alkyl, an aralkyl, an aryl, analkyl-cycloalkyl, a cycloalkyl, an alkyl-heterocycle, a heterocycle; andR⁶ and R⁷ are independently selected from hydrogen, an alkyl, an aryl, ahydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a halogen, anamino, an acyl, an acyloxy, an amido and nitro.
 2. The compound of claim1, wherein the compound is described by the structure:

wherein: Z² is a covalent bond, or a linking functional group; and R⁵ isselected from hydrogen and an alkyl.
 3. The compound of claim 1, whereinthe compound is described by the structure:

wherein: R¹ is selected from an aryl, an aralkyl, an alkyl-heterocycle,a heterocycle, a cycloalkyl and an alkyl-cycloalkyl; R² is hydrogen,alkyl or alkoxy; R³ is alkyl; R⁴ is alkyl, cycloalkyl, alkyl-cycloalkyl,heterocyclyl or alkyl-heterocyclyl; W¹ is —SO₂— or —C(═O)—; and W² is acovalent bond, —NH—, or —NHCO—.
 4. The compound of claim 3, wherein thecompound is described by the structure:

wherein: R¹ is —(CH₂)_(m)—R²⁰, where R²⁰ is an aryl, a cycloalkyl or aheterocycle and m is 0, 1 or 2; R² is an alkoxy; R³ is an alkyl; W² is acovalent bond, —NH—, or —NHCO—; n is 0, 1, 2 or 3; and R¹⁰ is acyclopropyl, a cyclopentyl, a cyclohexyl, a piperidinyl or apyrrolidinyl.
 5. The compound of claim 1 wherein R¹ or R²⁰ is describedby the structure:

wherein A is a 6-membered aryl, heteroaryl, heterocyclyl, or cycloalkylring, wherein Z¹¹-Z¹⁶ are independently selected from N. CR′, NR andCR′R″, where R is H, an alkyl, an aryl or a heterocycle; and R′ and R″are independently selected from hydrogen, an alkyl, an aryl, a hydroxy,an alkoxy, an aryloxy, a heterocycle, a cyano, a halogen, an amino, anacyl, an acyloxy, an amido, and a nitro.
 6. The compound of claim 1,wherein R² is lower alkoxy.
 7. The compound of claim 1, wherein R³ islower alkyl.
 8. The compound of claim 1, wherein the compound isdescribed by one of the following structures:

wherein R¹ is —(CH₂)_(m)—R²⁰, where R²⁰ is an aryl, a cycloalkyl or aheterocycle and m is 0, 1 or
 2. 9. The compound of claim 1, wherein R¹or R²⁰ is described by one of the following structures:

wherein R⁴⁴-R⁴⁶ are independently selected from hydrogen, an alkyl, anaryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, ahalogen, an amino, an acyl, an acyloxy, an amido, and a nitro; R′ ishydrogen, an alkyl, an aryl or a heterocycle; n² is 0, 1, 2 or 3, and n¹is 0, 1 or
 2. 10. The compound of claim 1, wherein the compound isdescribed by a structure of Table
 1. 11. An anti-infectivepharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable excipient.
 12. A method of inhibiting aPI4-kinase, the method comprising contacting a sample comprising thePI4-kinase with the compound of claim
 1. 13. The method of claim 12,wherein the PI4-kinase is a PI4-III kinase.
 14. The method of claim 13,wherein the PI4-III kinase is a PI4KIIIα- or PI4KIIIβ-kinase.
 15. Amethod of treating a subject for an infective disease condition, themethod comprising administering to the subject an effective amount ofthe compound of claim
 1. 16. The method of claim 15, wherein theinfective disease condition is caused by infection of a pathogensusceptible to PI4-kinase inhibition.
 17. The method of claim 16 whereinthe infective disease condition results from infection with a pathogenselected from the group consisting of HCV, rhinovirus (e.g., B or C). P.falciparum, ebola virus, francisella tularensis, hantavirus, vaccinia,smallpox. Japanese encephalitis virus, hepatitis A virus, and influenzavirus, PolioVirus, Enterovirus (e.g., A-D), West Nile Virus,cytomegalovirus, P. aeruginosa, and Dengue Virus (e.g., 1-4).
 18. Themethod of claim 17, wherein the pathogen is HCV.
 19. The method of claim16, wherein the compound has broad-spectrum activity against two or morepathogens.
 20. The method of claim 16, wherein the compound modulatesthe activity of a PIP-2-dependent process.
 21. The method of claim 16,wherein the pathogen is characterized by having a BAAPP domain thatinteracts with PIP-2, wherein the BAAPP domain is derived from NS5A orNS4B protein.
 22. A method of inhibiting viral infection, the methodcomprising contacting virus-infected cells with an effective dose of thecompound of claim 1 to inhibit viral replication.
 23. The method ofclaim 22, further comprising contacting the cells with a secondantiviral agent.
 24. The method of claim 22, wherein the compound isformulated to be targeted to the liver.